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| Status |
Public on Apr 01, 2024 |
| Title |
K562_U6DDR_Doxy_Rep3 |
| Sample type |
SRA |
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| Source name |
K562
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| Organism |
Homo sapiens |
| Characteristics |
cell line: K562
cell type: lymphoblast cells
genotype: U6DDR-sgRNA
treatment: Nucleofection
dsb induction: Cas12a
polymerase: Taq
description (number_of_normalized_reads): PE300
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| Treatment protocol |
G1/G0-arrested K562-iCas9-DDR cell lines were treated by 50 μM Palbociclib. The cycling and G1/G0-arrested cells were transduced with Cas12a plus DDR1-crRNA nucleoproteins to generated twined DSBs in different compound-treated conditions.
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| Growth protocol |
K562-iCas9-DDR cell line was cultured at 37 °C and 5% CO2 in RPMI supplemented with 10% (vol/vol) FBS, 50 U/mL penicillin/streptomycin, 2 mM L-glutamine , 1× MEM-NEAA, 1 mM sodium pyruvate , 50 μM 2-mercap- toethanol and 20 mM Hepes (pH 7.4).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was isolated using protocol described in Hu et al., 2016 Nature Protocols (PMC4895203)
DDR libraries were constructed through a two-step PCR process. In the initial PCR, outer layer primers (DDR-lib-F1 and DDR-lib-R1) were employed to amplify the regions of interest. Subsequently, in the second PCR, I5-DDR and I7-DDR primers were used to tag the libraries, which were then purified through electrophoresis and a gel-extraction kit.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
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| Data processing |
PCR-mediated template switching, involved the extraction and merging of barcodes from the paired-end sequencing library based on sequencing identification (ID). Paired barcodes underwent pairwise alignment and were compared with a reference barcode list to determine matched, switched, and randomly occurring errors.
The mapping of each paired library to the reference DNA template was executed using CRISPResso2 and stored in BAM files.
The ID and Cigar information were extracted and generated into TXT files, which were then merged with the matched file containing barcodes identified by ID.
Gene information was incorporated based on the sgRNA spacer sequence.
For the analysis of repair outcomes, it was assumed that the Double-Strand Breaks (DSB) occurred within ±10bps of the ideal break site. This implies that the deletion window must overlap with the DSB window, and all insertions must reside within this DSB window.
Assembly: hg19, pMCB320-DDR, pMCB320-RuDDR
Supplementary files format and content: tab delimited text files contain the pairwise alignment information including: the index, the barcode 1 (sequence_x), sequence identity (ID), length of barcode 1 (len_x), the barcode 2 or the spacer (sequence_y), length of barcode 2 (len_y), the reverse complement of barcode 2 (sequence_y_rcp) and the alignment score between barcode 1 and the barcode 2 (Align_score).
Supplementary files format and content: tab delimited text files contain the filtered, annotated mapping information including: the Index, the sequencing identity (ID), the barcode 1 (barcode), the barcode 2 or sgRNA spacer (seq_exp), the alignment score between barcode 1 and barcode 2 (Align_score), the gene name (Symbol), the mapping to a 362bp region of interest in the reference template (Cigar), the length of deletion (D_length), the position of deletion (D_location), the length of insertion (I_insertion), the position of insertion (I_location).
Library strategy: Dual-barcode Repair-Seq
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| Submission date |
Jan 13, 2024 |
| Last update date |
Apr 01, 2024 |
| Contact name |
Jinglong Wang |
| E-mail(s) |
biopioneering@gmail.com
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| Phone |
6507992088
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| Organization name |
Stanford University
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| Department |
Radiation Oncology
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| Street address |
1291 Welch Rd, CCSR Room 1240
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| City |
Stanford |
| State/province |
CA California |
| ZIP/Postal code |
94305 |
| Country |
USA |
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| Platform ID |
GPL15520 |
| Series (1) |
| GSE253191 |
Dual Barcode is Essential for the Specificity of DSB Repair Capture |
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| Relations |
| BioSample |
SAMN39430076 |
| SRA |
SRX23205688 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8016195_analyzed_U6DDR_Doxy_3_w_annotation.txt.gz |
560.6 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
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