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| Status |
Public on May 11, 2017 |
| Title |
Control sgRNA rep3 |
| Sample type |
SRA |
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| Source name |
Lymphoblastoid cell lines
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| Organism |
Homo sapiens |
| Characteristics |
cell line: GM12878
genotype/variation: Control sgRNA
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| Treatment protocol |
GM12878 with stable Streptococcus pyogenes Cas9 were established by lentiviral transduction using the pLentiCas9-Blast(Addgene plasmid #52962) and blasticidin selection. For all transductions, lentiviruses were made by 293T (obtained from ATCC) transfection. To make specific gene knockouts, control or sgRNA oligos against IRF2, IRF4, and BATF were synthesized by Integrated DNA Technologies, Inc. and cloned into the pLentiGuide-Puro vector (Addgene plasmid #52963). Puromycin selection (3μg/ml) was added 48 hours post transduction. Cells were collected for downstream analysis 3 days after puromycin selection. All sgRNAs were validated for their efficiency of gene knockout by western blot. Each biological group is performed in triplicates.
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| Growth protocol |
Gm12878 were cultured in GIBCO RPMI 1650 medium (Life Technologies) with 10% fetal calf serum (FCS) in a humidified chamber at 37 degrees Celcius with 5% CO2.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Prior to sample preparation for RNA-seq, dead cells were removed on day 3 after puromycin selection (day 5 after lentiviral transduction of sgRNAs) using Dead Cell Removal Kit (Miltenyi Biotec #130-090-101, San Diego, CA) following the manufacturer’s manual. Total RNAs were subsequently isolated using PureLink RNA Mini Kit (Thermo Fisher Scientific) following the manufacturer’s manual. An in-column DNA digestion step was included to remove any residual genomic DNA contamination.
To construct RNA-seq libraries, 500 ng total RNA was used for polyA mRNA-selection using NEBNext Poly(A) mRNA Magnetic Isolation Module (#E7490, New England Biolabs, Inc. Ipswich, MA), followed by library construction via NEBNext Ultra RNA Library Prep Kit for Illumina (#E7530, New England Biolabs, Inc., Ipswich, MA).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
BATF.sgRNA_vs_Control.sgRNA.txt
IRF2.sgRNA_vs_Control.sgRNA.txt
IRF4.sgRNA_vs_Control.sgRNA.txt
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| Data processing |
Adaptor-trimmed Illumina reads for each individual library were mapped back to the human GRCh37.83 transcriptome assembly using STAR v2.5.2b
featureCounts v1.5.1 was used to estimate the number of reads mapped to each contig. Only transcripts with at least 5 cumulative mapping counts were used in this analysis.
DESeq2 v1.14.1 was used to evaluate gene differential expression (DE)
Each DE analysis was composed of a pairwise comparison between experimental group and the control group. Differentially expressed genes were identified after a correction for false discovery rate (FDR 0.05).
Genome_build: GRCh37.83
Supplementary_files_format_and_content: tab-delimited text files include Log2FoldChange value (experimental v.s. control), p value and FDR corrected p value (padj). Separate tab-delimited text files are also provided for raw counts and DESeq2 normalized counts of genes, respectively
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| Submission date |
Jan 17, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Benjamin Gewurz |
| E-mail(s) |
bgewurz@bwh.harvard.edu
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| Organization name |
Brigham and Women's Hospital
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| Department |
Medicine
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| Street address |
181 Longwood Ave
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| City |
Boston |
| State/province |
Massachusetts |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE93681 |
CRISPR/Cas9 Screens Reveal Epstein-Barr virus Synthetic Lethal Targets |
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| Relations |
| BioSample |
SAMN06234869 |
| SRA |
SRX2496624 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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