|
| Status |
Public on May 24, 2023 |
| Title |
Jurkat-Lib3-Rep1 |
| Sample type |
SRA |
| |
|
| Source name |
Jurkat-Lib3-Rep1
|
| Organism |
Homo sapiens |
| Characteristics |
celi type: Jurkat-Cas9 cell
|
| Treatment protocol |
For screening in Jurkat-Cas9 and K562-cas9 cell line using “ON” and “OFF” library, 15 million cells were resuspended with 6ml RPMI in the presence of 8 µg/ml polybrene, and we transferred 1ml cell suspension into each well of 6-well plate. Predetermined volume of virus in the same 6-well format was added into each well to achieve a MOI of 5. After centrifugation at 600g for 2h at 32°C, 2ml pre-warmed RPMI with 15% FBS and 8 µg/ml polybrene was immediately added into each well of 6-well plate. For screening in H1-Cas9 cell line using “ON” and “OFF” library, the same number of cells should be plated on each matrigel-coated T175 flask 48 hours prior to transduction. When the monolayer cell density reaches to the optimal 50%-60% confluency, a T175 flask of cells were dissociated for cell counting (~20 million cells/T175). Calculated volume of virus was added into each T175 to achieve a MOI of 2 with 20ml mTeSR medium in the presence of 6 µg/ml polybrene. Cells were selected with puromycin (2.5-3 µg/ml) for 2d (Jurkat-cas9 and K562-cas9) or 3.5d (H1-cas9) at 36h post-transfection to remove uninfected cells. Cells were harvested at day 3.5 after transduction (day 5 for H1)
|
| Growth protocol |
HEK293T cell was grown in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gemini #900-108) and 1% penicillin/streptomycin (Gibco #15140-122). K562 and Jurkat cells were grown in RPMI 1640 (SIGMA #R8758) with 10% FBS (Gemini #900-108) and 1% penicillin/streptomycin. H1 cells were cultured on Matrigel (Corning)-coated plates with mTeSR medium (StemCell Technologies). Medium was changed daily. Cells were passaged every 3–4 days using ReLeSR (StemCell Technologies). K562 and Jurkat cells were harvested at day-3.5 post-transduction and H1 cells were collected at day-5.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA (gDNA) was isolated using DNeasy Blood & Tissue Kits according to the manufacturer’s protocol (Qiagen).
The region that was integrated with genomic DNA including both the gRNA, target sequence, and barcode was PCR amplified from genomic DNA for HTS. Primers for HTS were used to specifically amplify all of the gDNA using 25 µl NEBNext Ultra II Q5 Master Mix (New England Biolabs), 17 µl of gDNA (~2.5ug), 2.5 µl of primer mix at a final concentration of 250 nM, and 5.5 µl water. For each gDNA sample, PCR reactions were in the range from 30 to 60.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
Hlib4-S.R1
OnTarget-OffTarget.xlsx
DSB_Repair_Map.xlsx
Jurkat_integrated_DSB_repair_category.xlsx
|
| Data processing |
Library strategy: Amplicon Sequencing
Reads adapter were removed by cutadapt, then the reads were mapped to the library using bowtie2. oligo with synthesis error in the gRNA region were removed.
The repairing pattern was classified according to the sequecing reads and the library design.
Genome_build: custermized oligo library
Supplementary_files_format_and_content: excel
|
| |
|
| Submission date |
Aug 10, 2021 |
| Last update date |
May 24, 2023 |
| Contact name |
Lijia Ma |
| E-mail(s) |
malabdata@westlake.edu.cn
|
| Organization name |
Westlake university
|
| Department |
School of Life Sciences
|
| Lab |
Lijia Ma
|
| Street address |
Shilongshan Road No.18, Cloud Town, Xihu District
|
| City |
Hangzhou |
| State/province |
Zhejiang |
| ZIP/Postal code |
310024 |
| Country |
China |
| |
|
| Platform ID |
GPL24676 |
| Series (1) |
| GSE181774 |
Comprehensive profiling of activity and specificity of CRISPR/Cas9 under cellular environment by deep learning |
|
| Relations |
| BioSample |
SAMN20694867 |
| SRA |
SRX11705329 |
