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| Status |
Public on Dec 01, 2023 |
| Title |
Effector_T_3 |
| Sample type |
SRA |
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| Source name |
gut
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
transfection: pST-sgRNA
treatment: none
genotype: Rag1-/- NM
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| Treatment protocol |
Activated T cells were transfected with pST-sgRNA RV in the presence of 8 mg/mL polybrene during spin infection (2,000 g for 120 min at 33 ºC) and then rested for 3 days in medium containing 20 U/mL hIL-2 (Peprotech), 1 mg/mL a-TGFβ (BioXcell), 5 ng/mL mouse IL-7 (Sinobio), 1 mg/mL a-mouse IFNg (BioXcell) and 1 mg/mL a-mouse IL-4 (BioXcell). CD90.1 positive activated T cells were sorted and transferred into Rag1–/– recipient mice, with 300,000 cells for 1 recipient through i.v. tail. After 21 days, recipients were sacrificed and dissected to get intestinal tissues.
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| Growth protocol |
T cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES, 100 mM non-essential amino acids (NEAA), 50 U/mL penicillin, 50 mg/mL streptomycin, and 50 mM b-mercaptoethanol without further indication. For retroviral vector (RV) experiment, TCR transgenic naïve CD4+ T cells were sorted from a HH7-2tg Cas9 mouse, then activated under TH0 condition with plated coated a-mouse CD3e (0.25 mg/mL, BioXcell) and a-mouse CD28 (1 mg/mL, BioXcell) for 2 days, the medium was supplemented with 1 mg/mL a-mouse IFNg (BioXcell), 1 mg/mL a-mouse IL-4 (BioXcell), 20 U/mL hIL-2 (Peprotech) and 1 mg/ml a-TGFβ (BioXcell) to prevent TH1, TH2 or TH17 polarization.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Intestinal tissues were washed by PBS with 1 mM DTT for 10 min, followed by 5 mM EDTA for 20 min to remove epithelial cells, then treated by RPMI with collagenase (1 mg/mL collagenase; Roche), DNase I (100 μg/mL; Sigma), dispase (0.05 U/mL; Worthington) and 10% FBS at 37ºC for 60 min with constant mixing. Leukocytes were collected by 40-80% Percoll gradient. Inflammatory T cells were sorted with CD45.1+ and CD90.1+. Genomic DNA was extracted by the standard protocols with proteinase K treatment.
Genomic DNA was fragmented to be 300 bp in size by sonication. DNA fragments were applied to a Bst polymerase 3.0 (NEB) mediated one-round of liner primer extension with a biotinylated primer targeting the on-target site. After that, the excessive biotinylated primers were removed by AxyPrep Mag PCR Clean-Up beads (Axygen). DNA was denatured at 95℃ for 5 min, immediately chilled on ice for 3 min, and incubated with Dynabeads MyOne Streptavidin C1 (Thermo Fisher) for 2-4 hours. The C1 beads were thoroughly washed by 1x B&W buffer (5 mM Tris-HCl, pH 7.4; 1 M NaCl; 1 mM EDTA), 10 mM NaOH, 10 mM Tris-HCl, and then subjected to be ligated with a bridge adapter containing random molecular barcodes. After overnight incubation, the ligated DNA on C1 beads was thoroughly washed by 1x B&W buffer and 10 mM Tris-HCl, and tagged with Illumina adapter sequences. DNA ranging from 300-700 bp were recovered and sequenced
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
cells sorted by CD3+ CD4+ CD45.1+ TCR-Vb6+ CD62L+ CD44- CD25-, activated by a-CD3/CD28, a-IFNg, and a-IL-4 for 2days, and transfected with retrovirus with pST-sgRNA for 3days, CD90.1 positive activated T cells were sorted and transcfected into Rag1-/- recipient mice, after 21 days,recipients were sacrificed and inflammentory T cells from gut were sorted by CD90.1 and extract DNA
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| Data processing |
Illumina RTA software used for basecalling.
Both Illumina adapter sequences and ending low quality sequences (QC < 30) were trimmed by cutadapt (http://cutadapt.readthedocs.io/en/stable/); remaining reads shorter than 25bp were discarded.
The reported pipeline used in PEM-seq, termed superQ, was applied to perform reads alignment and translocation break point detection.
The reported pipeline, superQ, was applied to perform duplicates identification.
Break-site and neighbor ±5bp region was analyzed for indels; reads containing large deletions resulting from resection and rejoining in the break-site ±250kb region were also categorized as indels (I). Reads without any detected mutations around break point were identified as germline (G). Genome-wide translocation (T) was identified as described in PEM-seq.
Genome_build: mm10
Supplementary_files_format_and_content: tab files generated by superQ
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| Submission date |
Jul 11, 2021 |
| Last update date |
Dec 01, 2023 |
| Contact name |
Yang Liu |
| E-mail(s) |
liu.y@pku.edu.cn
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| Organization name |
School of life sciences, Peking University
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| Lab |
Jiazhi Hu lab
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| Street address |
Yiheyuan Road, No. 5
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| City |
Beijing |
| ZIP/Postal code |
100871 |
| Country |
China |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE179884 |
Chromomal translocations induced by CRISPR-Cas9 persist in expanded T cells |
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| Relations |
| BioSample |
SAMN20166428 |
| SRA |
SRX11407550 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5436502_Effector_T_3.txt.gz |
5.2 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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