Small RNA-Seq in epididymis of wild type and Alkbh3-/- mice
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE112871 | GEO | 2021/04/09
REPOSITORIES: GEO
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Project description:Total RNA (100 μg) were denatured at 70 °C for 5 min and separated by a 15% TBE-Urea gel with 20/100 or 10/60 oligo length standard ladder (Integrated DNA Technologies, Coralville, IA). After visualized by SYBR Gold (Ref. S11494 from Life Technologies), tRFs in gels were cut according to sizes and recovered by small RNA PAGE recovery kit (Zymo Research, Irvine, CA, USA). The recovered tRFs was used to establish the library by NEB small RNA library preparation kit (E7330S).
2021-04-21 | GSE113491 | GEO
Project description:Total RNA (100 μg) were denatured at 70 °C for 5 min and separated by a 15% TBE-Urea gel with 20/100 or 10/60 oligo length standard ladder (Integrated DNA Technologies, Coralville, IA). After visualized by SYBR Gold (Ref. S11494 from Life Technologies), tRFs (15~50 nt) in gels were cut according to sizes and recovered by small RNA PAGE recovery kit (Zymo Research, Irvine, CA, USA). The recovered tRFs was used to establish the library by NEB small RNA library preparation kit (E7330S).
2021-04-21 | GSE113490 | GEO
Project description:Total RNA (100 μg) were denatured at 70 °C for 5 min and separated by a 15% TBE-Urea gel with 20/100 or 10/60 oligo length standard ladder (Integrated DNA Technologies, Coralville, IA). After visualized by SYBR Gold (Ref. S11494 from Life Technologies), tRFs (15~50 nt) in gels were cut according to sizes and recovered by small RNA PAGE recovery kit (Zymo Research, Irvine, CA, USA). The recovered tRFs was used to establish the library by NEB small RNA library preparation kit (E7330S).
2021-04-09 | GSE112870 | GEO
Project description:The CLIP procedures were conducted using HeLa cells according to the previous study (Liu et al., 2016) with slight modifications. Briefly, 4 plates of HeLa cells in 15 cm dishes were transfected with pPB/ALKBH3 plasmid for 48 h to reach about 90% cell confluency. After washing twice with ice-cold PBS, cells were irradiated twice with 400 mJ/cm2 at 254 nm by Stratalinker on ice. Cells were lysed in high salt lysis buffer (300 mM NaCl, 0.2% NP-40, 20 mM Tris-HCl PH 7.6, 0.5 mM DTT, protease inhibitor cocktail (1 tablet/50 ml), and RNase inhibitor (1:200)) at 4°C for 30 min. Supernatant was collected after centrifuged at 17,000 g at 4°C for 15 min and further treated with 1 U RNase T1 for 15 min at 24 °C. After centrifugation and filtration through 0.22 µM filter, 10% supernatant was collected as input for further sequencing analysis. Anti-Flag M2 beads (Sigma-Aldrich, St. Louis, MO, 80 μl) were washed three times with lysis buffer and incubated with the filtered supernatant at 4°C for 4 hrs. After removing the flow through by magnetic rack, beads and input were further incubated with 10 U RNase T1 exactly for 8 min. Samples were treated with 95 µL commercial PNK buffer and 0.5 U PNK for 15 min at 37 °C. After treatments, final concentration of 100 µM ATP and 1 U PNK were added and incubated at 37 °C for another 30 min. The RNA/protein complexes were eluted by NuPAGE 1 × loading buffer and fractionated by neutral NuPAGE 4–12% bis-tris gel. After cutting the gel, RNAs were recovered by proteinase K digestion in proteinase K buffer, followed by phenol/chloroform extraction using glycogen as carrier. The RNA fragments were ligated to adapter and established library by NEB small RNA library preparation kit (E7330S).
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Project description:A total of 286 synthetic miRNAs that are commonly observed in circulation were synthesized by Integrated DNA Technologies (Coralville, IA).
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2021-08-12 | GSE181718 | GEO
Project description:We evaluated the effect of the small RNA library preparation method on 5' tRNA-halves and miRNA abundance in libraries prepared from serum RNA using three commercially available small RNA library preparation kits (TruSeq small RNA library preparation kit v2 (Illumina), TailorMix miRNA sample preparation kit v2 (Seqmatic) and the NEBNext Multiplex Small RNA library prep kit (New England Biolabs)). RNA isolated from 100 µl of serum collected from healthy mice was used as input for the preparation of a small RNA library in duplicate and libraries were single end sequenced.
2017-02-22 | GSE88914 | GEO
Project description:High-throughput RNA-sequencing has now become the gold standard method for whole-transcriptome gene expression analysis. It is widely used in a number of applications studying various transcriptomes of cells and tissues. It is also being increasingly considered for a number of clinical applications, including expression profiling for diagnostics or alternative transcripts detection. However, RNA sequencing can be challenging in some situations, for instance due to low input quantities or degraded RNA samples. Several protocols have been proposed to overcome some of these challenges, and many are available as commercial kits. Here we perform a comprehensive testing of three recent commercial technologies for RNA-seq library preparation (Truseq, Smarter and Smarter Ultra-Low) on human reference tissue preparations, for standard (1ug), low (100 and 10 ng) and ultra-low (< 1 ng) input quantities, and for mRNA and total RNA, stranded or unstranded. We analyze the results using read quality and alignments metrics, gene detection and differential gene expression metrics. Overall, we show that the Truseq kit performs well at 100 ng input quantity, while the Smarter kit shows degraded performances for 100 and 10 ng input quantities, and that the Smarter Ultra-Low kit performs quite well for input quantities < 1 ng. All the results are discussed in details, and we provide guidelines for the selection of a RNA-seq library preparation kits by biologists.
2019-05-10 | GSE124198 | GEO
Project description:These RNAseq data are from germinating cyp79B2, cyp79B3, myb28, myb29 (qko), cyp79B2, cyp79B3 (cyp) and myb28 myb29 (myb) mutant seeds. Col-0 genotype was used as the Arabidopsis' WT reference ecotype and all mutant seeds are from the Col-0 genetic background. Samples were sown on agarose 0,8%, supplemented or not by nitrate KNO3 10Mm or potassium thiocyanate KSCN 8µM. RNA isolation was performed using NucleoSpin® RNA Plant and Fungi kit. RNA quantification was obtained with a NanoDrop ND-100 (NanoDrop Technologies, DE, USA) and the RNA Integrity was measured with a 4100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). cDNA library construction and sequencing were performed with DNBseq™ technology at Beijing Genomics Institute BGI. FastQC was used to check read quality, mapping analysis and transcript quantiifcation were performed using a quasi-mapping alignment from Salmon.
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Project description:Pseudomonas extremaustralis, an Antarctic bacterium, was grown at low oxygen conditions for 24h and then exposed to S-Nitrosoglutathione (GSNO)100 µM for 1 h. RNA from treated and control samples was isolated using the Trizol method. RNA quality was analyzed on the Agilent Bioanalyzer and rRNA depletion was performed using the RiboZERO kit (Illumina). Samples were validated using an Agilent 2100 Bioanalyzer (Agilent Technologies). Libraries were prepared with TruSeq RNA Library Prep Kit v2 (Illumina) and sequenced with the Illumina NextSeq 550 platform with a single-end protocol.
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