ChIP-seq of circadian transcription factors in MLL-AF9 leukemia cell lines
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Carl de Boer
PROVIDER: E-GEOD-70686 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Commercially available histone antibodies were analyzed using peptide microarrays to assess specificity. For two H3K27me3 antibodies from different vendors, we performed ChIP-seq in WT and EED-null murine ES cells to assess specificity. Cell lines were generated from e3.5 blastocysts of EED fl/fl animals and transfected with plasmid CAG-CRE-ERT2. Null cells were generated by single cell clones from tamoxifen-treated parent cell line. Analysis of the specificity of two H3K27me3 antibodies using WT and EED (Embryonic Ectoderm Develepment)-null (mutants lacking H3K27me3) ES cells. The goal was to compare WT and KO for each antibody with the expectation that if the antibodies were specific, there would remain few peaks and little signal in the knockout. We sequenced 2 replicates of each condition (3 for WT input) and after comparing replicates, we merged data, called peaks, and calculated signal in bins centered on the wild type H3K27me3 peaks. As little to no signal remained either by direct comparison of peaks or looking at 'metagenes' of the peaks, we concluded that the antibodies were specific. Antibodies: Abcam H3K27me3 (ab6002, lot# GR130002-1) Upstate H3K27me3 (07-449, lot# 2455635)
2015-06-21 | E-GEOD-66902 | biostudies-arrayexpress
Project description:Our primary objective was to characterize the amount of variation in transcript abundance among individual flies with identical genotypes. We also wanted to determine which analysis methods would be optimal for RNA-Seq data. To meet these objectives, we performed transcriptional profiling of whole adult individuals from 16 Drosophila Genetic Reference Panel (DGRP) lines. We quantified differential expression among genotypes, environments, and sexes. We randomly chose 16 DGRP lines for this experiment: DGRP-93, DGRP-229, DGRP-320, DGRP-352, DGRP-370, DGRP-563, DGRP-630, DGRP-703, DGRP-761, DGRP-787, DGRP-790, DGRP-804, DGRP-812, DGRP-822, DGRP-850, and DGRP-900. We collected 8 virgin male and 8 virgin female flies from the 16 DGRP genotypes in three replicated environments to produce RNA sequence profiles. We controlled the environmental conditions in the following ways. We seeded the fly cultures with 5 male and 5 female parents. We reared the progeny in a single incubator on standard Drosophila food (http://flystocks.bio.indiana.edu/Fly_Work/media-recipes/bloomfood.htm) at 25°C, 60% humidity, and a 12:12-hour light:dark cycle. We collected and maintained male and female virgins at 20 flies to a same-sex vial for four days prior to RNA extraction to control for social exposure. Flies were frozen for RNA extraction at the same circadian time (1:00 pm) in 96-well plates. PolyA RNA stranded libraries were prepared by modifying an existing protocol. ERCC (External RNA Controls Consortium, SRM2374, beta version, pools 78A/78B) sequences were added during the library preparation as a control. For some samples >1 library was generated to check technical variation. We performed multiplexed single-end 76 bp sequencing on an Illumina HiSeq2000. Reads were mapped to FlyBase release 5 version 57 and release 6 version 01 of the Drosophila melanogaster genome and the ERCC sequences. Mapped reads were counted at the gene level.
2015-12-21 | E-GEOD-60314 | biostudies-arrayexpress
Project description:We performed an RNA-seq analysis on FACS-sorted CD4+ memory T cells (Tmem, CD4+CD25-CD45RA-) stimulated in vitro with anti-CD3/CD28 coated beads (1:5), IL-2 (100 U/mL) in the presence of a TNF-alpha inhibitor (Etanercept, ETN, 5 μg/mL) or recombinant human TNF-alpha (rhTNF-alpha, 50 ng/mL) treatment. The cells were cultured for three days at 37°C and 5% CO2. On day three Tmem were lysed (Buffer RLT supplemented with DTT, QIAGEN) and RNA extraction was performed (RNeasy Plus Micro kit), followed by sample preparation with TruSeq (polyA) mRNA kits (Illumina), RNA sequencing (carried out by an Illumina HiSeq2500), and the alignment of trimmed fastQ files to GRCh38 human reference genome. We also performed a Quality control (QC). This was done using the tool FastQC FastQC/0.11.3-Java-1.7.0_80. QC metrics were calculated for the aligned reads using Picard-tools picard/1.130-Java-1.7.0_80, CollectRnaSeqMetrics, MarkDuplicates, CollectInsertSize-Metrics and SAMtools/1.2-foss-2015b flagstat. Finally, we carried out a Differential expression gene (DEG) analysis using DESeq2, in order to evaluate the impact of ETN and rhTNF-alpha on Transcriptome profiling of stimulated CD4+ Tmem. Principal Component Analysis (PCA) was carried out to visualise the samples given their entire transcriptome and any remaining batch effects.
2017-12-15 | E-MTAB-5622 | biostudies-arrayexpress
Project description:Background: Recently we characterised TRB1, a protein from a single-myb-histone family, as a structural and functional component of telomeres in Arabidopsis thaliana. TRB proteins, besides their ability to bind specifically telomeric DNA using their N-terminally positioned myb-like domain of the same type as in human TRF1 or TRF2 shelterin proteins, possess also histone-like domain which is involved in protein-protein interactions e.g., with POT1b. We set to investigate the genome-wide localization pattern of TRB1 to reveal its preferential mode of binding to chromatin in vivo and its potential functional roles in the genome-wide context. Results: Our results demonstrate that in addition to its roles at telomeres, TRB1 is preferentially associated with promoter regions of genes involved in ribosome biogenesis. This preference coincides with a frequent occurrence of telobox motifs in the upstream regions of genes in this category, but is not restricted to the telobox presence. Conclusions: We conclude that TRB1, in addition to its preferential telomeric localization, shows a specific genome-wide distribution pattern suggesting its role in regulation of genes involved in biogenesis of translational machinery. TRB1 DNA-binding activity investigated in 2 biological replicates (2 immunoprecipitation approaches) with controls; with technical replicates.
2015-12-21 | E-GEOD-69431 | biostudies-arrayexpress
Project description:To test the role of Mpe1 in transcription termination we inserted a mini-auxin induced degron (mAID) at the C-terminal end of the endogenous MPE1 locus in the yeast Saccharomyces cerevisiae. We treated Mpe1-mAID cells (JRY101) or wild type cells (WT, YMK728) with 1 mM auxin for 30 minutes and labeled the nascent RNA with 4-thiouracil (4tU) for 6 minutes. The nascent RNA fraction was prepared by biotinylating the 4tU-labeled RNA followed purification with streptavidin beads. Nascent and total RNA fractions were depleted of ribosomal RNA and strand-specific libraries prepared. Libraries were single-end sequenced using an Ilummina HiSeq 4000 instrument.
2021-12-31 | E-MTAB-10820 | biostudies-arrayexpress
Project description:In mammals, the peptide hormone vasopressin controls renal water excretion, largely through its actions in the renal collecting duct to regulate the molecular water channel aquaporin-2 (AQP2). There are two modes of regulation: 1) short-term regulation by membrane trafficking; and 2) long-term regulation involving vasopressin-induced changes in the abundance of the aquaporin-2 protein. Defects in the long-term regulation have been implicated in multiple water balance disorders. With the objetive to identify genes that are transcriptionally regulated in response to vasopressin in cultured mouse collecting duct cells (mpkCCD), we carried out both RNA-seq to measure all transcripts (n=9) and ChIP-seq for RNA polymerase II (Polr2a) binding along the genome (n=3). Observations were made both after 24-hr treatment with the vasopressin analog dDAVP and with vehicle. Protocol:Confluent monolayers mpkCCD cells were washed in ice-cold 1X PBS (Ca+2/Mg+2-free) followed by cross-linking in 1.11% formaldehyde in Covaris Fixing Buffer (5.5 ml) for 5min at room temperature with gentle rocking (PN 520075, Covaris, Woburn, Massachusetts). Cross-linking was terminated by the addition of 0.3 ml of 1X Covaris Quenching Buffer with rocking. The buffer solution was aspirated and 1X ice-cold PBS was added to each plate. Cells were scraped into 15ml conical tubes, spun down (200 x g, 5 min), and washed twice with PBS. After the second wash, cell pellets were resuspended in 10 ml of 1X Covaris Lysis Buffer for 10 min at 4o C with rocking. Nuclei were pelleted by spinning at 1,700 x g for 5 min at 4o C. Nuclear pellets was resuspended in the 1X Covaris Wash Buffer and incubated for 10 mins at 4o C with rocking. Samples were spun at 1,700 x g for 5 min at 4o C. Nuclear pellets were washed twice with the Covaris Non-ionic Shearing Buffer and spun at 1,700 x g for 5 min at 4o C. Nuclei were resuspended in Covaris Non-ionic Shearing Buffer (Maximum of 1-3x 10^7cells per ml of buffer). Shearing. Two 20 ul aliquots of the resuspended pellets were removed prior to shearing to be used as subsequent unsheared controls for immunoblotting and agarose gel electrophoresis. The remaining samples were transferred to AFA tubes (TC12 x 12 mm) and volumes adjusted to 1 ml (V)). Samples were sheared using a Covaris S2 SonoLAB Single system for two minutes (Duty cycle: 5%; Intensity: 4; cycle: 200). Total nuclear protein concentration of each sample in mcg/ml (R) was determined with the BCA Protein Assay Kit (Thermo Scientific, Rockford, IL). To test shearing efficiency, a 20 ul aliquot from each tube was removed after shearing to test the efficacy of shearing. The aliquots, including the unsheared sample, were incubated with 100 ul 1X TE buffer, 10 ul 10% SDS, and RNAse A (10 ug/ul, Cell Signaling Technology, Danvers, Massachusetts) for 30 mins at 37o C. 1 ul proteinase K (20 ug/ul) was added and the samples were incubated overnight at 65o C. The DNA was purified using DNA Clean & Concentrator columns (Catalog #: D4013, Zymo Research, Irvine, CA) and ran on an E-gel EX 2% agarose (Invitrogen, Carlsbad, CA). Target fragment sizes for chromatin immunoprecipitation were in the range of 150-700 bp in length with average fragment size of 300 bp. Testing epitope integrity (immunoblotting). Total protein content was determined with the BCA Protein Assay Kit (Thermo Scientific, Rockford, IL). Samples were then diluted with sample buffer (5X Laemmli buffer) at 20% (v/v) of the entire sample volume. Proteins were resolved by SDS-PAGE (4-20% polyacrylamide gels, Criterion, Bio-Rad, Hercules, CA) and transferred electrophoretically onto nitrocellulose membranes as previously described (Hwang, 2010). Membranes were probed with anti-RNA polymerase II (Catalog #: MMS-126R, Covance). The antibody was used at a 1:500 dilution (anti-RNA polymerase II) (in Odyssey blocking buffer containing 0.1% Tween 20) overnight at 4°C. After 1-h incubation with secondary antibody (Li-Cor Biosciences 680 anti-rabbit immunoglobulin G or 800 anti-mouse immunoglobulin G; Lincoln, NE) at 1:5000 dilution, sites of antibody-antigen reaction were detected using an Odyssey infrared imager (Li-Cor). Chromatin immunoprecipitation. 10 ul of sample equivalent to 2% input was removed prior to IP. Chromatin Immunoprecipitation was performed using 30-90 ug of sheared chromatin in 500 uL of 1X Covaris Non-ionic Shearing Buffer with 10 μl anti-RNA polymerase II antibodies. Samples were incubated overnight at 4°C with rotation, followed by addition of ChIP Grade Protein G Magnet Beads (Catalog #: 9006, Cell Signaling Technology) and incubation for 2 h at 4o C with rotation. The beads were washed a total of four times, including three low salt washes followed by one high salt wash for 5 mins/wash at 4o C (SimpleChIP protocol #9003, Cell Signaling Technology). Immunoprecipitated chromatin was eluted from the beads with 150 ul of 1X ChIP Elution Buffer for 30 mins at 65o C in a thermomixer. To reverse crosslinks, 6 ul of 5M NaCl and 2 ul of 20 ug/ul Proteinase K were added to the chromatin followed by incubation at 65o C overnight. DNA was purified according to SimpleChIP protocol #9003, Cell Signaling Technology. q-PCR. To check the success of the chromatin immunoprecipitation, input and ChIPâ??ed DNA samples were amplified with SYBR Green PCR Master Mix (Applied Biosystems) on a 7900HT Fast Real-Time PCR System. Primer sets corresponding to Hox9a and actin genes were used as positive controls for both histone and RNA polymerase II IPâ??s. (see Supplementary Table X for primer sequences). Library construction, amplification and sequencing. Loaded fixed volume of total eluate. Library construction was performed on a NuGEN instrument using the protocol and reagents outlined in the Ovation SP Ultralow Library Systems kit. Libraries were then enriched via PCR. After amplification, DNA was measured. A fixed amount of the total measured DNA was used for sequencing in an Illumina Hiseq2000 instrument to obtain single read data. High-throughput sequencing and read mapping. Libraries for Illumina sequencing of the samples were prepared using the the protocol and reagents outlined in the Ovation SP Ultralow Library Systems kit , NuGen Mondrian SP+ (NuGEN, San Carlos, CA) . Samples were sequenced using an Illumina HiSeq2000, obtaining 1- by 50-bp reads. We combine RNA-seq and ChIP-seq to identify genes whose mRNA abundances change in tandem with changes in RNA polymerase II binding, as a mean to identify genes that are transcriptionally regulated in response to vasopressin. In addition, the pattern of RNA polymerase II binding along the gene provides important information about regulatory mechanisms. We identify two populations of genes regulated by vasopressin in mpkCCD cells, viz. 1) those with increases in transcript abundance and increases in RNA polymerase II binding throughout the gene body and; 2) genes that show increases in RNA polymerase II binding in the promotor proximal region but do not produce increases in transcript level. The former group is restricted to 35 genes and includes Aqp2. The latter group is made up of the majority of expressed genes and is presumably due to a widespread increase in transcriptional initiation without proportionate release of pausing.
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