|
| Status |
Public on Jul 08, 2024 |
| Title |
Day7_mKate2_Neg_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
4D6
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: 4D6
cell type: thymic epithelial cells
genotype: Cas9-Blast, FuGW-G5p-mKate2, EGFP-P2A-Gal4DBD-AireCTT
treatment: Lentiviral library transduction, puromycin selection, Dox treatment, sorting
time: Day7
phenotype: mKate2 negative
|
| Treatment protocol |
The engineered 4D6 cells were transduced with lentiviral Human Brunello CRISPR knockout pooled sgRNA library [a gift from David Root and John Doench (Addgene #73178-LV)7] at a MOI of 0.4, aiming for 500-fold representation of each sgRNA. Library transduced cells were selected under 1 mg/ml puromycin for 2 days and further expanded for another 7 or 10 days. Cells were treated with 1 µg/ml Dox 24 hrs prior to sorting to induce EGFP-P2A-Gal4DBD-AireCTT expression, and then top-5% and bottom-5% of the population were sorted based on mKate2/EGFP ratios on the SH800S Cell Sorter (Sony Biotechnology).
|
| Growth protocol |
4D6 cells were maintained in RPMI supplemented with 10% FBS, 1% L-glutamine
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted using the DNeasy Blood and Tissue Kit (QIAGEN, #69504) and cleaned using the OneStep PCR Inhibitor Removal Kit (Zymo Research, #D6030).
Sequencing libraries were generated by PCR amplification as previously described (Doench JG et al, 2016), pooled at an equal molar concentration, purified using the MinElute Reaction Cleanup Kit (Qiagen, #28204) to enrich for the 350-360bp amplicons
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
Demultiplexed sequencing reads were trimmed using Cutadapt (v2.5) to remove vector-derived sequences, yielding only 20 bp sequences corresponding to sgRNAs.
The statistical analysis of sgRNA enrichment was performed using MAGeCK-VISPR (v0.5.6) with the "MAGeCK-RRA" experimental configuration and visualized using the MAGeCKFlute R package (v2.0.0).
Assembly: Human Brunello CRISPR knockout pooled sgRNA library
Supplementary files format and content: tab-delimited txt file includes CRISPR screening scores
Library strategy: Targeted genomic sequencing
|
| |
|
| Submission date |
Sep 22, 2023 |
| Last update date |
Jul 08, 2024 |
| Contact name |
Qianxia Zhang |
| E-mail(s) |
qianxia.zhang@childrens.harvard.edu
|
| Phone |
9018345955
|
| Organization name |
Boston Children's Hospital
|
| Street address |
3 Blackfan Circle, RM 3117.16
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL20301 |
| Series (2) |
| GSE243823 |
Mechanism for controlled assembly of transcriptional condensates by Aire [CRISPR] |
| GSE243825 |
Mechanism for controlled assembly of transcriptional condensates by Aire |
|
| Relations |
| BioSample |
SAMN37515205 |
| SRA |
SRX21860245 |
