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Sample GSM5436498 Query DataSets for GSM5436498
Status Public on Dec 01, 2023
Title Naïve_T_primer_control
Sample type SRA
 
Source name spleen
Organism Mus musculus
Characteristics strain: C57BL/6
transfection: none
treatment: none
genotype: HH7-tg Cas9 CD45.1
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.
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.
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
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina HiSeq 2000
 
Description cells sorted by CD3+ CD4+ CD45.1+ TCR-Vb6+ CD62L+ CD44- CD25-, and extract DNA
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
 
Submission date Jul 11, 2021
Last update date Dec 01, 2023
Contact name Yang Liu
E-mail(s) liu.y@pku.edu.cn
Organization name School of life sciences, Peking University
Lab Jiazhi Hu lab
Street address Yiheyuan Road, No. 5
City Beijing
ZIP/Postal code 100871
Country China
 
Platform ID GPL13112
Series (1)
GSE179884 Chromomal translocations induced by CRISPR-Cas9 persist in expanded T cells
Relations
BioSample SAMN20166432
SRA SRX11407546

Supplementary file Size Download File type/resource
GSM5436498_Naive_T_primer_control.txt.gz 21.8 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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