|
| Status |
Public on Dec 06, 2023 |
| Title |
CD40L-KO rep2 |
| Sample type |
SRA |
| |
|
| Source name |
PBMCs from healthy donors
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: PBMCs
sample: CAST-seq derived amplicons from gDNA
treatment: Cas9-treated (two RNPs targeting CD40L)
|
| Treatment protocol |
Electroporation of CAR T cells was carried out using the 4D-Nucleofector, kit P3 (Lonza) . The S.p. Cas9 was purchased from Integrated DNA Technologies. The gRNA was purchased from Biolegio. 48h before electroporation the cells were transduced with a lentivirus to generate the CAR T cells. 24h post-electroporation the IL-2 concentration was reduced in the media medium until reached 100 U/ml. 8 days po-electoporation the cells were checked for phynotiping and genotyping
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| Growth protocol |
The T cells and the gene-edited CAR T cells T cells were cultivated in RPMI-1640 medium supplemented with 10% FCS, 1% P/S, 10 mM HEPES, 100 U/ml IL-2 , 25 U/ml IL-7 and 50 U/ml IL-15
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was isolated from cultured cells after 8 days using the DNeasy blood & tissue kit (Qiagen) according to the manufacturer’s instructions.
Library construction was essentially performed as described by Turchiano et. al (Cell Stem Cell, 2021). Briefly, gDNA was fragmented by enzymatic digestion using the NEBNext® Ultra™ II FS DNA Library Prep (NEB) to obtain average fragment lengths of 200-350 bp. The DNA was end repaired and a protruding 3’-A nucleotide was added according to the manufacturer’s instructions in order to enable the ligation of a linker sequence with a protruding 3’-T. After linker ligation, fragments were purified using the QIAquick PCR Purification Kit (NEB). The region of interes (on-target site) was amplified in two rounds of PCR using Q5® Hot Start High-Fidelity DNA Polymerase (NEB). PCR conditions were: 20 cycles at 95°C for 15 s, 63°C (first reaction) or 68°C (second reaction) for 20 s, 72°C for 20 s. The first reaction was performed with primers complementary to the linker sequence and to a sequence in close proximity to the on-target site (bait primer). Two decoy primers were introduced to reduce full-length amplification of the fragments which contain the on-target sequence without a chromosomal aberration event. The second PCR utilized nested primers to reduce the amount of mis-amplified fragments and to introduce a binding platform for barcoding primers. DNA fragments from the second PCR with a size of 200-500bp were subsequently isolated using the QIAquick® Gel Extraction Kit (Qiagen) and barcoded.
Third PCR introduced the barcoded Illumina adapter for sequencing (NEBNext® Multiplex Oligos for Illumina; Index Primers Sets 1-4). Sequencing was outsourced to service NGS provider Genewiz (part of Azenta Life Sciences) on where it was performed on Illumina HiSeq or NovaSeq devices collecting 2x150 bp paired-end reads.
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| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
CAST-seq performed at the CD40L locus on sample treated with two RNP complexes (Cas9) targeting CD40L
CD40L-KO_FINAL.xlsx
|
| Data processing |
Mate paired reads were merged using BBmerge from BBmap (v38.76) software. BBmap was also used for filtering and trimming as follow: merged reads containing the designer nuclease target site were filtered-in, whereas PCR mispriming products reads were filtered-out. Linker sequences, Illumina adapter sequences, targeted elongation sequence and bad quality reads were trimmed.
Selected reads were aligned to the human genome GRCh38 (hg38) using Bowtie2 (v2.3.4.2) and the very-sensitive preset parameters. Aligned reads with good mapping quality (MAPQ >15) were selected. The aligned BAM file was converted into bed file using bedtools (v2.27.1).
Translocated sites were identified using the CAST-Seq pipeline with default parameters, as described on https://github.com/AG-Boerries/CAST-Seq
Assembly: hg38
Supplementary files format and content: matrix table of translocation sites detected with CAST-Seq pipeline. It includes the genomic coordinates of translocated sites, read coverage, alignment score against gRNA, translocation event classification and nearest genes annotation.
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| |
|
| Submission date |
Sep 19, 2023 |
| Last update date |
Dec 06, 2023 |
| Contact name |
Geoffroy Andrieux |
| Organization name |
University clinics Freiburg
|
| Street address |
Breisacherstr 153
|
| City |
Freiburg |
| ZIP/Postal code |
79110 |
| Country |
Germany |
| |
|
| Platform ID |
GPL20301 |
| Series (1) |
| GSE243587 |
Cell-based models of ‘Cytokine Release Syndrome’ endorse CD40L and GM-CSF knockout in CAR T cells as mitigation strategy |
|
| Relations |
| BioSample |
SAMN37472102 |
| SRA |
SRX21827851 |
