A8760cf1-404b-4685-a85b-d15055cf03fc - samples
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PROVIDER: EGAD00001010881 | EGA |
REPOSITORIES: EGA
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Project description:The RRBS libraries of the genomic DNA from the 521 tissue samples were constructed following the standard RRBS protocol. 100-200 ng of intact genomic DNA in the volume of 21.5 µl was used as input material. Restriction digestion was done with 2.5 µl 10xCutSmart buffer and 1 µl MspI (NEB) for 18 h at 37 oC and 20 min at 65 oC. 0.5 µl 10xCutSmart buffer, 0.3 µl dACGTP mixture (100 mM dATP, 10 mM dCTP, 10 mM dGTP), 1 µl Klenow (exo-, 5U/µl, NEB) and 2.6 µl RT-PCR water, 0.6 µl 50 mM DTT (ThermoFisher) was added to the mixture for end repair and A-overhang addition with the program 30 oC for 20 min, 37 oC for 1 h and 75 oC for 20 min. Adapter ligation was then performed with 1 µl 10xThermoFisher HC T4 ligase buffer, 0.4 µl 100 mM ATP (ThermoFisher), 0.2 µl 50 mM DTT, 1 µl ThermoFisher HC T4 DNA ligase (30 Weiss Unit/µl), 30 ng home-made duplex UMI adapter with all the cytosines methylated (protocol adopted from Kennedy et al.) at 16 oC for 20 h and 65 oC for 20 min. Bisulfite conversion of the adapter-ligated product was carried out with QIAGEN EpiTect plus DNA bisulfite kit following their protocol for two rounds of conversion. The converted product was purified with Qiagen MinElute spin column and eluted with 20 µl RT-PCR water. PCR amplification was done using the NEBNext Multiplex Oligos for Illumina (2.5 µl of universal and index primer each) and 25 µl KAPA HiFi HotStart Uracil+ ReadyMix (Roche) with the following cycling conditions: 98 oC for 45 s, 9 cycles of 98 oC for 15 s, 60 oC for 30 s and 72 oC for 30 s, followed by a final extension at 72 oC for 5 min. The PCR product was purified with 1x AmpureXP beads and eluted with 30 µl EB buffer. DNA concentration was measured by Qubit 1xdsDNA HS assay. 5% TBE-UREA PAGE and bioanalyzer assay was performed as quality control on each library before sequencing.
| EGAD00001010880 | EGA
Project description:ATAC-seq data were generated for a number of selected patients to investigate changes in enhancer and promoter regions. ATAC-seq was essentially carried out as described in31. Briefly, prior to transposition the viability of the cells was assessed and 1 × 106 cells were treated in culture medium with DNase I (Sigma) at a final concentration of 200 U ml−1 for 30 minutes at 37 °C. After Dnase I treatment, cells were washed twice with ice-cold PBS, and cell viability and the corresponding cell count were assessed. 5 × 104 cells were aliquoted into a new tube and spun down at 500 × g for 5 minutes at 4 °C, before the supernatant was discarded completely. The cell pellet was resuspended in 50 µl of ATAC-RSB buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2) containing 0.1% NP-40, 0.1% Tween-20, and 1% Digitonin (Promega), and was incubated on ice for 3 minutes to lyse the cells. Lysis was washed out with 1 ml of ATAC-RSB buffer containing 0.1% Tween-20. Nuclei were pelleted at 500 × g for 10 minutes at 4 °C. The supernatant was discarded carefully and the cell pellet was resuspended in 50 µl of transposition mixture (25 µl 2× tagment DNA buffer, 2.5 µl transposase (100 nM final; Illumina), 16.5 µl PBS, 0.5 µl 1% digitonin, 0.5 µl 10% Tween-20, 5 µl H2O) by pipetting up and down six times. The reaction was incubated at 37 °C for 30 minutes with mixing before the DNA was purified using the Monarch PCR & DNA Cleanup Kit (NEB) according to the manufacturer’s instructions. Purified DNA was eluted in 20 µl elution buffer (EB) and 10 µl purified sample was objected to a ten-cycle PCR amplification using Nextera i7- and i5-index primers (Illumina). Purification and size selection of the amplified DNA were carried out with Agencourt AMPure XP beads. For purification the ratio of sample to beads was set to 1:1.8, whereas for size selection the ratio was set to 1:0.55. Purified samples were eluted in 15 µl of EB. Quality and concentration of the generated ATAC libraries were analyzed using the High Sensitivity D1000 ScreenTape Kit (Agilent) and libraries were sequenced paired-end on a NovaSeq (Illumina).
ATAC-seq reads were aligned to the human reference genome build hg19 with bowtie2
| EGAD00001007583 | EGA
Project description:Escherichia coli strain MG1655 was grown to mid-log phase in defined anaerobic media. One sample was treated with 100 µM CORM-3, the other sample was a control. The volume (175 ml), temperature (37oC) and stirring (200 rpm) were constant. After 15 min of exposure to CO-RM, samples were taken from treated and control cells, harvested into ice cold phenol ethanol (187 µl phenol, 3.56 ml ethanol) to stabilize RNA, and total RNA was purified using Qiagenâs RNeasy Mini kit as recommended by suppliers. RNA was quantified using a BioPhotometer (Eppendorf). Biological experiments were carried out four times, and a dye swap performed for each experiment, providing two technical repeats for each of the four biological repeats. Cells were grown in defined minimal medium (anaerobic). Final concentrations are: glycerol (54 mM), K2HPO4 (23 mM), KH2HPO4 (7.3 mM), NH4Cl (18.7 mM), CaCl2 (69 µM), K2SO4 (15 mM), sodium fumarate (50 mM), Luria Broth (5%) and can amino acids (0.1%). Trace elements were added at 10 ml per l; final concentrations are: 134 µM EDTA, 31 µM FeCl3, 6.15 µM ZnO, CuCl2 0.57 µM, CoNo3 0.34 µM, 1.6 µM H3BO3, 1 µM ammonium molybdenate and 1 µM sodium selenite. MgCl2 was added at 1 ml per l. All chemicals were of AnalaR grade of purity or higher. E. coli strain MG1655 was grown in mini fermenter vessels in 175 ml of the above defined minimal media (anaerobic). The vessel was continually sparged with N2 to ensure anaerobiosis. CORM-3 was added at a final concentration of 100 µM, chosen because it causes only a slight reduction in the growth rate. Cells were grown to mid-log (OD600 of 0.4) before addition of CO-RM. For RNA extraction, 30 ml was harvested into ice cold phenol ethanol (187 µl phenol, 3.56 ml ethanol) to stabilize RNA, and total RNA was purified using Qiagenâs RNeasy Mini kit as recommended by suppliers. RNA was quantified using a BioPhotometer (Eppendorf). Biological experiments were carried out four times, and a dye swap performed for each experiment, providing two technical repeats for each of the four biological repeats. Equal quantities of RNA from control and CO-RM-treated cells were labelled by using nucleotide analogues of dCTP containing either Cy3 or Cy5 fluorescent dyes (Perkin Elmer). For each microarray slide, one sample was labelled with Cy3-dCTP, while the other sample was labelled with Cy5-dCTP. RNA (approximately 9 micrograms) was annealed to 4.5 micrograms pd(N)6 random hexamers (Amersham Biosciences) by heating at 65 oC for 10 min, followed by 10 min at 22 oC and cooling on ice for 2 min. This was supplemented with 6 microlitres of 5à First-strand buffer (Invitrogen), 2.5 microlitres dNTP mixture (5 mM each dATP, dTTP, dGTP and 2 mM dCTP), 3 microlitres 0.1 M DTT, 2 microlitres 1 mM Cy3 or Cy5 (Perkin Elmer) and 1.5 microlitre Superscript III reverse transcriptase (200 U) (Invitrogen). This was incubated at 42 oC for 3 hours. The reaction was terminated by the addition of NaOH (7.5 microlitres of 1M) and HCl (7.5 microlitres of 1M) before the addition of TE buffer (300 microlitres). Labelled cDNA was purified using a Qiaquick PCR purification kit (Qiagen). The Cy3-dCTP-labelled sample was mixed with the Cy5-dCTP-labelled sample, vacuum dried and re-suspended in 120 microlitres salt-based hybridisation buffer (supplied with the microarray slides). This was heated at 95 oC for 3 min then cooled on ice for 3 min. The mixture was pipetted onto a microarray slide, sealed with a GeneFrame and coverslip in a hybridization chamber and incubated for 18 h at 42 °C. Following hybridization, microarray slides (minus GeneFrame and coverslip) were washed in a series of pre-warmed (37 °C) SSC buffers for 5 min each at 37oC: 2à SSC/0.1 % SDS, 1à SSC, 0.2à SSC and 0.01à SSC. Microarray slides were dried by centrifugation at 1500 à g for 2 min before scanning. Slides were scanned on an Affymetrix 428 scanner. The average signal intensity and local background correction were obtained using a commercially available software package from Biodiscovery, Inc (Imagene, version 4.0 and GeneSight, version 3.5). Spots automatically flagged as bad, negative or poor in the Imagene software were removed before the statistical analysis was carried out in GeneSight. The mean values from each channel were log2 transformed and normalised using the Lowess method to remove intensity-dependent effects in the log2(ratios) values. The Cy3/Cy5 fluorescent ratios were calculated from the normalized values. Biological experiments (i.e. a comparison of control and plus CO-RM cells) were carried out four times, and a dye swap performed for each experiment, providing two technical repeats for each of the four biological repeats. Data from the independent experiments were combined. Genes that were differentially expressed ⥠twofold and displayed and P value of < 0.05 (as determined by a t test) were defined as being statistically significantly differentially transcribed.
2010-06-24 | E-GEOD-12878 | biostudies-arrayexpress
Project description:Escherichia coli strain MG1655 was grown to mid-log phase in defined aerobic media. One sample was treated with 30 µM CORM-3, the other sample was a control. The volume (30 ml), temperature (37oC) and shaking (200 rpm) were constant. After 15 min of exposure to CO-RM, samples were taken from treated and control cells, harvested into ice cold phenol ethanol (187 µl phenol, 3.56 ml ethanol) to stabilize RNA, and total RNA was purified using Qiagenâs RNeasy Mini kit as recommended by suppliers. RNA was quantified using a BioPhotometer (Eppendorf). Biological experiments were carried out four times, and a dye swap performed for each experiment, providing two technical repeats for each of the four biological repeats. Cells were grown in defined minimal medium (aerobic). Final concentrations are: glycerol (54 mM), K2HPO4 (23 mM), KH2HPO4 (7.3 mM), NH4Cl (18.7 mM), CaCl2 (69 µM), and K2SO4 (15 mM). Trace elements were added at 10 ml per l; final concentrations are: 134 µM EDTA, 31 µM FeCl3, 6.15 µM ZnO, CuCl2 0.57 µM, CoNo3 0.34 µM, 1.6 µM H3BO3, 1 µM ammonium molybdenate and 1 µM sodium selenite. MgCl2 was added at 1 ml per l. All chemicals were of AnalaR grade of purity or higher. E. coli strain MG1655 was grown in 250 ml side arm flasks in 30 ml of the above defined minimal media (aerobic). CORM-3 was added at a final concentration of 30 µM, chosen because it causes only a slight reduction in the growth rate. Cells were grown to mid-log (Klett values of 30-40) before addition of CO-RM. The total 30 ml volume was harvested into ice cold phenol ethanol (187 µl phenol, 3.56 ml ethanol) to stabilize RNA, and total RNA was purified using Qiagenâs RNeasy Mini kit as recommended by suppliers. RNA was quantified using a BioPhotometer (Eppendorf). Biological experiments were carried out four times, and a dye swap performed for each experiment, providing two technical repeats for each of the four biological repeats. Equal quantities of RNA from control and CO-RM-treated cells were labelled by using nucleotide analogues of dCTP containing either Cy3 or Cy5 fluorescent dyes (Perkin Elmer). For each microarray slide, one sample was labelled with Cy3-dCTP, while the other sample was labelled with Cy5-dCTP. RNA (approximately 9 micrograms) was annealed to 4.5 micrograms pd(N)6 random hexamers (Amersham Biosciences) by heating at 65 oC for 10 min, followed by 10 min at 22 oC and cooling on ice for 2 min. This was supplemented with 6 microlitres of 5à First-strand buffer (Invitrogen), 2.5 microlitres dNTP mixture (5 mM each dATP, dTTP, dGTP and 2 mM dCTP), 3 microlitres 0.1 M DTT, 2 microlitres 1 mM Cy3 or Cy5 (Perkin Elmer) and 1.5 microlitre Superscript III reverse transcriptase (200 U) (Invitrogen). This was incubated at 42 oC for 3 hours. The reaction was terminated by the addition of NaOH (7.5 microlitres of 1M) and HCl (7.5 microlitres of 1M) before the addition of TE buffer (300 microlitres). Labelled cDNA was purified using a Qiaquick PCR purification kit (Qiagen). The Cy3-dCTP-labelled sample was mixed with the Cy5-dCTP-labelled sample, vacuum dried and re-suspended in 120 microlitres salt-based hybridisation buffer (supplied with the microarray slides). This was heated at 95 oC for 3 min then cooled on ice for 3 min. The mixture was pipetted onto a microarray slide, sealed with a GeneFrame and coverslip in a hybridization chamber and incubated for 18 h at 42 °C. Following hybridization, microarray slides (minus GeneFrame and coverslip) were washed in a series of pre-warmed (37 °C) SSC buffers for 5 min each at 37oC: 2à SSC/0.1 % SDS, 1à SSC, 0.2à SSC and 0.01à SSC. Microarray slides were dried by centrifugation at 1500 à g for 2 min before scanning. Slides were scanned on an Affymetrix 428 scanner. The average signal intensity and local background correction were obtained using a commercially available software package from Biodiscovery, Inc (Imagene, version 4.0 and GeneSight, version 3.5). Spots automatically flagged as bad, negative or poor in the Imagene software were removed before the statistical analysis was carried out in GeneSight. The mean values from each channel were log2 transformed and normalised using the Lowess method to remove intensity-dependent effects in the log2(ratios) values. The Cy3/Cy5 fluorescent ratios were calculated from the normalized values. Biological experiments (i.e. a comparison of control and plus CO-RM cells) were carried out four times, and a dye swap performed for each experiment, providing two technical repeats for each of the four biological repeats. Data from the independent experiments were combined. Genes that were differentially expressed ⥠twofold and displayed and P value of < 0.05 (as determined by a t test) were defined as being statistically significantly differentially transcribed.
2010-06-24 | E-GEOD-12877 | biostudies-arrayexpress
Project description:YEAST STRAIN:,A genetically modified Saccharomyces cerevisiae yeast strain with PMI40 knockout (MATa URA3 TRP1 LEU2 his3- 1 MAL2-8C SUC2 PMI40::HIS3). The parent strain was CEN.PK113-7A (MATa URA3 TRP1 LEU2 his3- 1 MAL2-8C SUC2) obtained from Euroscarf (9).,CULTIVATION MEDIUM:,The mineral medium used for the bioreactor cultivations was based on the composition described by Verduyn and co-workers (11). The medium consisted of 5 g/l (NH4)2SO4 (Sigma-Aldrich, USA), 3 g/l KH2PO4 (Fluka, Swizerland) and 0.5 g/l MgSO4 à 7 H2O (Sigma-Aldrich, USA), as well as 1 ml/l trace element and 1 ml/l vitamin solution. The composition of the trace element solution was 15 g/l EDTA, 4.5 g/l ZnSO4 à 7 H2O, 0.84 g/l MnCl2 à H2O, 0.3 g/l CuSO4 à 5 H2O, 3 g/l FeSO4 à 7 H2O (Riedel-de-Haën AG, Germany), 0.1 g/l KI, 0.3 g/l CoCl3 à 6 H2O, 1 g/l H3BO3, 4.5 g/l CaCl2 à 2 H2O (Merck, Germany), and 0.4 g/l Na2MoO4 à 2 H2O (BDH Laboratory Supplies, UK). The composition of the vitamin solution was 0.05 g/l D-Biotin, 1 g/l Ca-pantothenate, 1 g/l Nicotinic acid, 25 g/l myo-inositol, 1 g/l Thiamine-HCl, 1 g/l Pyridoxine HCl, and 0.2 g/l p-aminobenzoic acid (Sigma-Aldrich, USA).,The media in each cultivation contained initially 15 g/L D-glucose and a varying amount of D-mannose (0.75 g/L, 1.0 g/L, 1.5 g/L, 3.0 g/L, or 5.0 g/L).,CULTIVATIONS AND SAMPLING:,Bioreactor cultivations were performed in 3.2 l Braun Biostat CT2-DCU 3 instruments (B. Braun Biotech International GmbH, Germany). The cultivation temperature was +30 °C, agitation speed 1000 rpm, initial cultivation volume 2.0 l, airflow 1.0 l/min, and pH 5.0.,In each cultivation, samples for the measurement of gene expression levels were taken 6 hours after inoculation.,MEASUREMENT OF GENE EXPRESSION LEVELS:, ,0.9 mg CDW of cells were quenched in cold (-40 °C) methanol (45%) and centrifuged (1 min, +4 °C, 16100 G). The pellets were suspended in 400 µl of breaking buffer (20 mM Tris-HCl pH 7.4, 100 mM KCl, 2 mM MgCl2, 2mM DTT), 400 µl 1:1 phenol-chloroform, and 5 µl 20% SDS. Equal volume of glass beads (diameter 0.40-0.60 mm, B.Braun Biotech International, Germany) were added and samples were shaken in a bead beater (Mini-BeadBeater-8, BioSpec Products, USA) three times 1 minute at +4 °C. The mixtures were centrifuged (15 min, +4 °C, 16100 G in 5415 R, Eppendorf AG, Germany) and supernatants were recovered. The total RNA was purified from the supernatants using the QIAGEN RNeasy Mini kit (Qiagen, USA) according to manufacturers instructions. After this step the double-stranded cDNA was synthesized from 15 µg of total RNA with SuperScript Double-Stranded cDNA synthesis kit (Invitrogen, USA) using a T7-oligo(dT) primer. Biotin-labeled cRNA was then synthesized employing an Enzo High Yield RNA Transcript Labeling Kit (Enzo Life Science, USA). The biotin-labeled cRNA was heat-fragmented and biotinylated cRNAs were hybridized to Affymetrix YG-S98 microarrays in Affymetrix Hybridization Oven 640. Washing and staining of arrays were performed using the GeneChip Fluidics Station 450 and scanning with the Affymetrix GeneChip Scanner 3000. Acquisition and quantification of array images were performed using the Affymetrix GeneChip Operating System 1.0. The signals were scaled into target intensity of 100. All samples were assayed in triplicates.
2007-07-08 | E-GEOD-1938 | biostudies-arrayexpress
Project description:Cells were washed with cold PBS twice and scraped from the plates, then centrifuged at 1000 rpm for 5 min to pellet cells. Washed cells were lysed with 400 L of cell lysis buffer (10 mM Tris-HCl pH7.5, 150 mM NaCl, 0.15% NP-40 and proteinase inhibitor cocktail) and incubated on ice for 10 min. The suspension was carefully add at the top of sucrose buffer (10 mM Tris-HCl pH7.5, 150 mM NaCl, 24% sucrose), and centrifuged at 3200g for 10 min to collect nuclear pellets. Pellets were resuspended in 250 L of Glycerol buffer (20 mM Tris-HCl pH7.5, 75 mM NaCl, 0.5 mM EDTA pH8.0, 50% glycerol), then then immediately add 250 μl Nuclear lysis buffer (10 mM Tris-HCl pH7.5, 300 mM NaCl, 7.5 mM MgCl2, 0.2 mM EDTA pH8.0, 1% NP-40, 1M urea). Mix by vortexing for 4 s and incubate on ice for 2 min. Centrifuge the lysate at 13,000 × g for 2 min to precipitate the chromatin–RNA complex. Briefly rinse the chromatin pellets with PBS-EDTA. RNA was extracted with Trizol reagent. RNA libraries were prepared according to manual of SMARTer smRNA-Seq Kit for Illumina (Clontech, 635031).
2019-10-15 | GSE134103 | GEO
Project description:Male mice on a homogenous C57BL6/J genetic background were sacrificed at 11 to 13 weeks of age by cervical dislocation. Brain, kidneys and liver were immediately harvested, flash frozen in liquid nitrogen and stored at -80°C until use. Animal handling was in accordance with guidelines approved by the European Molecular Biology Laboratory (EMBL). Organ sections (thickness 30 µm) were prepared in a Cryostat (Leica Biosystems, Leica CM3050 S) set to -20 °C and deposited onto SuperFrost glass slides (Carl Roth, H880); the glass slides were pre-cooled to -20°C in the cryostat. ~10 sections were placed on a glass slide, and a total of around 150 sections were prepared for each sample per mouse. Tissue sections on glass slides were then transferred onto metal plates placed on dry ice. The tissue sections were exposed or not to 1 J/cm2 of 254 nm UV light in a XL 1500 UV Spectrolinker (Spectronics Corporation) and subjected to eRIC to determine the RNA-bound proteome. eRIC eluates obtained from the respective organs of two mice were combined per independent experiment. The analyses were conducted in duplicate for each organ studied. For the no crosslink controls, organ sections from four mice were pooled to generate one unique no crosslink eRIC sample per organ studied.
2023-03-02 | PXD032113 | Pride
Project description:Flag-YBX1 overexpressed T24 cells pellets were resuspended with 2 volume of lysis buffer (150 mM KCl, 10 mM HEPES pH 7.6, 2 mM EDTA, 0.5% NP-40, 0.5 mM DTT, 1:100 protease inhibitor cocktail, 400 U/ml RNase inhibitor), and incubated at 4 °C for 30 min with rotation. Then the lysate was centrifuged at 15 000 g for 20 min. Before incubating the lysate with Flag beads, 100ul were taken as input. The anti-Flag M2 magnetic beads (Sigma, 10 μl per mg lysate) were washed with NT2 buffer (200 mM NaCl, 50 mM HEPES pH 7.6, 2 mM EDTA, 0.05% NP-40, 0.5 mM DTT, 200 U/ml RNase inhibitor) four times. Cell lysate was mixed with M2 beads and incubated at 4 °C for 4 h with rotation. The beads were washed two times with 1 ml ice-cold NT2 buffer. Then the beads were subject to Micrococal nuclease (NEB) digestion (1:1 000 000 dilution) for 8 min at 37 °C. The beads were cooled on ice immediately for 5 min and washed two times with 1 ml ice-cold 1× PNK+EGTA buffer (50 mM Tris-HCl pH 7.5, 20 mM EDTA, 0.05% NP-40, 200 U/ml RNase inhibitor) and two times with 1 ml ice-cold 1× PK buffer (50 mM NaCl, 100 mM Tris-HCl pH 7.5, 10 mM EDTA, 0.2% SDS, 200 U/ml RNase inhibitor). Then the beads were digested with 200 μl pre-heated (20 min at 50 °C) Proteinase K and RNAs were extracted with an equal volume of Acid-Phenol: Chloroform, pH 4.5 (Ambion). The RNAs were subjected to rRNA removal and Bisseq. The libraries were sequenced on the Illumina HiSeq X-Ten platform at Novogene (Tianjin, CA) with paired-end 150 bp read length.The m5C sites were called using meRanCall from meRanTK (FDR < 0.01).
2019-07-02 | GSE133625 | GEO
Project description:Escherichia coli strain MG1655 was grown in parallel chemostat cultures in GGM. In one chemostat the culture was fed adequate Zn; in the other, measures were taken to eliminate Zn from the chemostat vessel and culture medium. Culture volume (120 ml), temperature (37 oC) and stirring speed (437 rpm) were maintained. Steady state values for pH and OD600 were 6.9 and 0.6, respectively. After 50 hours, samples from the adequate Zn and Zn-limited chemostats were harvested into RNAprotect and total RNA was purified using Qiagenâ??s RNeasy Mini kit (using the supplierâ??s protocol) prior to use in microarray analysis. Biological experiments (i.e. a comparison of adequate versus low Zn in chemostat culture) were carried out three times, and a dye swap performed for each experiment, providing two technical repeats for each of the three biological repeats. Cells were grown in glycerol-glycerophosphate medium (GGM). Final concentrations in GGM are: MES (40.0 mM), NH4Cl (18.7 mM), KCl (13.4 mM),beta-glycerophosphate (7.64 mM), glycerol (5.00 mM), K2SO4 (4.99 mM), MgCl2 (1.00 mM), EDTA (134 microM), CaCl2.2H2O (68.0 microM), FeCl3.6H2O (18.5 microM), ZnO (6.14 microM), H3BO3 (1.62 microM), CuCl2.2H2O (587 nM), CoNO3.6H2O (344 nM), and (NH4)6Mo7O24.4H2O (80.9 nM) in MilliQ water (Millipore). Bulk elements (MES, NH4Cl, KCl, K2SO4 and glycerol in MilliQ water at pH 7.4 (batch growth) or 7.6 (continuous culture)) were passed through a column containing Chelex-100 ion exchange resin (Bio-Rad) to remove contaminating cations. Trace elements (with or without Zn as necessary) and a CaCl2 solution were then added to give the final concentrations shown above prior to autoclaving. After autoclaving, MgCl2 and beta-glycerophosphate were added at the final concentrations shown above. All chemicals were of AnalaR grade of purity or higher. Chelex-100 was packed into a Bio-Rad Glass Econo-column (approximately 120 mm Ã? 25 mm) that had previously been soaked in 3.5% nitric acid for 5 d. E. coli strain MG1655 was grown in parallel custom-built chemostats made entirely of non-metal parts. One chemostat was fed medium that contained â??adequateâ?? Zn (that normally found in GGM), whilst the other contained no added Zn and had been actively depleted of Zn by use of the special precautions listed here. Glass growth vessels and flow-back traps were soaked extensively (approximately two months) in 10% nitric acid before being rinsed thoroughly in MilliQ water. Vent filters (Vent Acro 50 from VWR) were connected to the vessel using PTFE tubing. Metal-free pipette tips were used (MAXYMum Recovery Filter Tips from Axygen). Culture volume was maintained at 120 ml using an overflow weir in the chemostat vessel. The dilution rate (and hence the specific growth rate) was 0.1 h-1. The vessel was inoculated using one of the side-arms. Flasks were placed on KMO 2 Basic IKA-Werke stirrers (arbitrary setting: 400). Stirring speed was calibrated (437 rpm) and matched between vessels using a handheld laser tachometer (Compact Instruments Ltd). Temperature was kept constant at 37°C. Twice daily, a 2-ml sample was taken and used to test the pH (which stayed constant at pH 6.9), OD600 (0.6 at steady state), glycerol limitation, and contamination on nutrient agar plates. The â??Zn-freeâ?? chemostat was inoculated with cells that had been sub-cultured in Zn-free medium to deplete internal stores of Zn. A 0.25 ml aliquot of a saturated culture of strain MG1655 grown in LB was centrifuged and the pellet used to inoculate 5 ml of GGM that was incubated overnight at 37°C with shaking. A 2.4 ml (i.e. 2% of chemostat volume) aliquot of this was then used to inoculate the chemostat. The â??adequate Znâ?? chemostat was inoculated with cells treated in essentially the same way but grown in GGM containing an â??adequateâ?? level of Zn. The two cultures (+/- Zn) used to inoculate the chemostats had OD600 readings within 2.5% of each other. Chemostats were grown for 50 h to allow five culture volumes to pass through the vessel and allow an apparent (pseudo-)steady state to be reached. At this point, 10 ml samples were removed from the chemostats and total RNA was stabilised using RNAprotect (Qiagen). Total RNA was purified using Qiagenâ??s RNeasy mini kit. Equal quantities of RNA from adequate Zn and Zn-limited chemostats were labeld by using nucleotide analogues of dCTP containing either Cy3 or Cy5 fluorescent dyes (Perkin Elmer). For each microarray slide, one sample was labeld with Cy3-dCTP, while the other sample was labeld with Cy5-dCTP. RNA (15-20 micrograms) was annealed to 9 micrograms pd(N)6 random hexamers (Amersham Biosciences) by heating at 65°C for 10 min, followed by 10 min at 22°C. This was supplemented with 4 microlitres of 5Ã? First-strand buffer (Invitrogen), 2 microlitres dNTP mixture (5 mM each dATP, dTTP, dGTP and 2 mM dCTP), 2 microlitres 0.1 M DTT, 2 microlitres 1 mM Cy3 or Cy5 (Perkin Elmer) and 1 microlitre Superscript II reverse transcriptase (200 U) (Invitrogen). This was incubated at 42°C for 2 hours. The reaction was terminated by the addition of NaOH (5 microlitres of 1M) and HCl (5 microlitres of 1M) before the addition of TE buffer (200 microlitres). Labelled cDNA was purified using a Qiaquick PCR purification kit (Qiagen). The Cy3-dCTP-labeld sample was mixed with the Cy5-dCTP-labeld sample and re-suspended in 120 microlitres salt-based hybridisation buffer (supplied with the microarray slides). This was heated at 95°C for 3 min then cooled on ice for 3 min. The mixture was pipetted onto a microarray slide, sealed with a GeneFrame and coverslip in a hybridization chamber and incubated for 18 h at 42 °C. Following hybridization, microarray slides (minus GeneFrame and coverslip) were washed in a series of pre-warmed (37 °C) SSC buffers for 5 min each at 37°C: 1Ã? SSC/0.1 % SDS, 1Ã? SSC, 0.2Ã? SSC and 0.01Ã? SSC. Microarray slides were dried by centrifugation at 500 Ã? g for 2 min before scanning. Slides were scanned on an Affymetrix 428 scanner. The average signal intensity and local background correction were obtained using a commercially available software package from Biodiscovery, Inc (Imagene, version 4.0 and GeneSight, version 3.5). Spots automatically flagged as bad, negative or poor in the Imagene software were removed before the statistical analysis was carried out in GeneSight. The mean values from each channel were log2 transformed and normalised using the Lowess method to remove intensity-dependent effects in the log2(ratios) values. The Cy3/Cy5 or Cy5/Cy3 (test/reference) fluorescent ratios were calculated from the normalized values. Biological experiments (i.e. a comparison of adequate versus low Zn in chemostat culture) were carried out three times, and a dye swap performed for each experiment, providing two technical repeats for each of the three biological repeats. Data from the independent experiments were combined. Data from the independent experiments were combined. Genes that were differentially expressed â?¥ twofold and displayed and P value of < 0.05 (as determined by a t-test) were defined as being statistically significantly differentially transcribed.
2010-06-25 | E-GEOD-11894 | biostudies-arrayexpress
Project description:Purpose: Investigation of DDX41 chromatin binding sites in U2OS cells Method: Expression of DDX41-GFP was induced for 48 hours before crosslinking using 1µg/ml docycycline. Control cells were not induced with doxycline. Cells were mildly crosslinked using 1% FA for 2 min at RT. Concanavalin A-coated beads were activated in Binding Buffer (20 mM Hepes-KOH pH 7.9, 10 mM KCl, 1mM CaCl2, 1 mM MnCl2).1*10^6 U2OS cells were washed twice with Wash Buffer (Hepes-NaOH pH7.5, 150 mM NaCl, 0.5 mM Spermidine, 1 mM protease inhibitor) at room temperature and afterwards immobilized on the activated beads in 1 ml Wash Buffer. Cells were permeabilized with 0.05% digitonin in Wash buffer for 4 min at RT. 1 µg Nanobody-GFP-MNase (in-house protein production, PMID:25362362) binding was performed at 4°C for 30 min in 0.05% digitonin-Wash buffer. After 2x washing, the MNase was activated by adding 3mM CaCl2 on ice for 30 min. 2x Stop Buffer (68 µl 5M NaCl, 40 µl 0.5 M EDTA, 20 µl 0.2 M EGTA, 10 µl 5% digitonin, 5 µl 10mg/ml RnaseA, )) was mixed with the samples to stop the reaction. Chromatin fragments were released by incubating the samples for 20 minutes at 37°C and centrifugation at 16.000×g for 10 minutes at 4°C. Supernatants were incubated at 70°C in the presence of 0.1% SDS and 5 µg proteinase K. Before library preparation, the DNA was recovered by phenol-chloroform extraction Results: We succesfully mapped DDX41 binding sites on cromatin in U2OS cells. DDX41 preferentially binds in the promoter region of genes.
2021-11-17 | GSE183083 | GEO