|
| Status |
Public on Nov 27, 2020 |
| Title |
Bko2 |
| Sample type |
SRA |
| |
|
| Source name |
human embryonic stem cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: H9
cell type: Embryonic stem cells
time point: Day 17
genotype/variation: pooled mutant cells
|
| Treatment protocol |
A total of 10 million PAX6-tdTomato/iCas9 hPSCs were transduced with each virus library at a multiplicity of infection (MOI) of 0.3, resulting in infection of 30% of the cells and hence leading to a 50-fold coverage of the sgRNA library size. After culturing at 37 ℃ for about 1.5 h, the transduced cells were plated on 6 six-well plates of MEF layer. At 24 h after transduction, cells were treated with puromycin (0.5 μg/ml) for 4 days to eliminate non-infected cells. Then doxycycline (Dox) were added to 5 six-well plates for 5 days, that one plate left without Dox treatment was as control. The cells with or without Dox treatment were passaged at a ratio of 1:5. Another 7 days later, the cells were digested with preheated trypsin to create a single cell suspension and tdTomato-positive cells were collected by FASC sorting (BD FACSVerse™ System, BD Biosciences). The wide-type ESCs and infected iCas9 ESCs without Dox treatment were as control cells.
|
| Growth protocol |
hPSCs were cultured in medium (DMEM/F12, KOSR, NEAA, β-Me, FGF2). hPSCs were cultured at 37 ℃ and 5% CO2 on feeder layer growth-arrested mouse embryonic fibroblasts (MEFs) in standard hESC growth medium, composed of DMEM/F12 with 20% Knockout Serum Replacement (KOSR),1x L-Glutamine,1x nonessential amino acids(NEAA), 0.1 mM β-mercaptoethanol and 4 ng ml-1 basic fibroblast growth factor (bFGF). Cells were passaged every 5 days with dispase digestion.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA of sorted cells was extracted with quickExtract DNA Extraction Solution (Epicentre, QE09050) and the sgRNAs were amplified by two-step PCR method.
For the second PCR, illumine adaptors and barcode were attached to samples. Amplicons from the second PCR were gel-extracted, quantified, mixed and sequenced using pair-ended 150bp sequencing protocol (Illumina Hiseq-x-ten system).
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
HiSeq X Ten |
| |
|
| Description |
processed data file: B_rawcounts.txt
|
| Data processing |
Library strategy: DNA sequencing
The sequencing reads of sgRNAs from different samples were first identified by barcode using cutadapt (version 1.11) with parameters –u (43, -87 for R1; 73, -57 for R2).
Bowtie2-build function of Bowtie2 (version 2.3.4.3) (Langmead and Salzberg 2012) was applied on the sgRNA sequences of GeCKO library to generate Burrows-Wheeler index. The sgRNA sequences were then retrieved and counted by aligning processed reads of each sample to the sgRNA library using Bowtie2 with default parameters and only the reads with unique alignment were reported.
The MAGeCK algorithm was used to estimate the statistical significant (using a negative binomial test) of enrichment for each gRNA in the KO-screened group compared to control group.
Genome_build: The sgRNAs sequences were from GeCKO library and could be download from
Supplementary_files_format_and_content: *_rawcounts.txt: Tab-delimited text files include raw counts.
|
| |
|
| Submission date |
Jun 06, 2019 |
| Last update date |
Nov 27, 2020 |
| Contact name |
yanhua du |
| E-mail(s) |
[email protected]
|
| Organization name |
Shanghai Jiaotong University School of Medicine
|
| Department |
Shanghai Institute of Immunology
|
| Street address |
227 South Chongqing Road, Huangpu District
|
| City |
Shanghai |
| ZIP/Postal code |
200025 |
| Country |
China |
| |
|
| Platform ID |
GPL20795 |
| Series (1) |
| GSE132309 |
Genome-scale CRISPR Screen Reveals Essential Biological Process Determining Human Lineage Specification |
|
| Relations |
| BioSample |
SAMN11969727 |
| SRA |
SRX5982465 |
