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| Status |
Public on Sep 15, 2014 |
| Title |
macroH2A1 KD replicate 3 |
| Sample type |
SRA |
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| Source name |
IMR90_macroH2A1 KD
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| Organism |
Homo sapiens |
| Characteristics |
cell line: IMR90
cell type: human primary lung fibroblasts
genotype/variation: expressing shRNA directed against macroH2A1
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| Growth protocol |
IMR90 (ATCC, CCL-186) primary human fetal lung fibroblast cells were cultured in MEM supplemented with 10% fetal bovine serum.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Tripure Isolation Reagent (Roche).
RNA samples were treated with RNase-free DNaseI and purified by using the RNeasy Mini Kit (QIAGEN). The resulting RNA was depleted of ribosomal RNA by using Ribo-Zero rRNA Removal Kit (Human/Mouse/Rat) (Epicentre). The rRNA-depleted RNA was used to synthesize the first strand cDNA using SuperScript® III First-Strand Synthesis System (Invitrogen) according to the manufacturers instructions. Subsequently, the second strand was generated by using E. coli DNA ligase (Invitrogen), E. coli DNA polymerase (Invitrogen), and dUTP, dCTP, dATP, and dGTP set (10 µmol each, Promega), in the Second Strand Buffer (Invitrogen). Then the cDNA were sheared with Covaris to roughly 300bp. After fragmentation, samples were purified with MinElute columns (Qiagen). And the protruding 3’ and 5’ ends of the fragments were repaired using T4 DNA Polymerase (NEB) and T4 DNA Polynucleotide kinase (NEB) in T4 DNA ligase buffer (NEB). Next, dA ends were generated by employing Klenow Fragment (3’->5’ exo-, NEB), in Buffer 2 (NEB). Adapter ligation reactions were then set up using indexed adapters and Quick Ligase (NEB). The products were used as the template for UNG (Fermentas) treatment and PCR amplification using Phusion High-Fidelity DNA Polymerase (NEB). The libraries were visualized on 2% E-Gel® General Purpose Agarose Gels (Invitrogen). The barcoded libraries were sequenced on a single lane of a HiSeq2500 (Illumina).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Sample 6
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| Data processing |
Base calls performed using CASAVA version 1.7
The sequencing reads were trimmed of barcodes and aligned to the human genome (hg19) using GSNAP version 2012-07-12
The reads were then binned into genes (Ensemble ens67) using HTSeq version v0.5.3p3 (http://www-huber.embl.de/users/anders/HTSeq/doc/index.html).
Identification of differential gene expression for protein coding genes with at least 20 reads across all replicates was performed with DESeq (version 1.10.1)
Genome_build: hg19
Supplementary_files_format_and_content: processed data files include uniquely mapping read counts for each gene
Supplementary_files_format_and_content: DESeq output consists of tab separated values for baseMean, baseMeanA(Luc KD), baseMeanB(macroH2A1 KD), foldChange, log2FoldChange, pval, padj.
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| Submission date |
Feb 10, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Matthew Jon Gamble |
| E-mail(s) |
matthew.gamble@einstein.yu.edu
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| Organization name |
Albert Einstein College of Medicine
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| Department |
Molecular Pharmacology
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| Lab |
Gamble Lab
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| Street address |
1300 Morris Park Ave / Golding 203
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| City |
Bronx |
| State/province |
NY |
| ZIP/Postal code |
10461 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE54846 |
RNA-seq from control and macroH2A1-depleted IMR90 primary human lung fibroblasts |
| GSE54847 |
Exploring the role of macroH2A1 in transcription regulation in IMR90 primary human lung fibroblasts with RNA-seq and ChIP-seq |
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| Relations |
| BioSample |
SAMN02639668 |
| SRA |
SRX469067 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1325079_m1-3.BD0YGGACXX.lane_6_P0_I6.hg19.htseq-union.out.txt.gz |
183.5 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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