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Sample GSM5436499 Query DataSets for GSM5436499
Status Public on Dec 01, 2023
Title Activated_T
Sample type SRA
Source name spleen
Organism Mus musculus
Characteristics strain: C57BL/6
transfection: pST-sgRNA
treatment: Activated by a-CD3/CD28, a-IFNg, and a-IL-4
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-, 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 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 (; 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
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
BioSample SAMN20166431
SRA SRX11407547

Supplementary file Size Download File type/resource
GSM5436499_Activated_T.txt.gz 3.0 Mb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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