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| Status |
Public on Jul 04, 2023 |
| Title |
CAST-seq on sample treated with two RNPs [UNC-u3d4-2] |
| Sample type |
SRA |
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| Source name |
HSPCs from bone marrow
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| Organism |
Mus musculus |
| Characteristics |
cell type: HSPCs
sample type: CAST-seq derived amplicons from gDNA
genotype: Jinx; C57BL/6J-Unc13dJinx/Mmucd
treatment: Cas9+sgRNAs u3 and d4-treated
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| Treatment protocol |
Lineage-negative (Lin-) HSCs (Miltenyi Biotec, 130-090-858) from femur and tibia-derived BM were stimulated for 3h in StemSpan SFEM II medium (StemCell Technologies) supplemented with L-glutamine (20 mM, Gibco), mSCF, mTPO and hIGF2 (20 ng/ml each) plus hIGF2 and mIL-3 (10 ng/ml each, Peprotech) before nucleofection (Lonza 4D nucleofector, P4 kit, program CA137) with two times 3µg Cas9 (IDT) complexed at a 1:3 molar ratio with sgRNAs (BioLegio). After approximately 20h of culture cells were harvested and their genomic DNA subjected to CAST-Seq.
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| Growth protocol |
Cells were cultured at 37°C and 5% CO2. Lineage-negative (Lin-) HSCs (Miltenyi Biotec, 130-090-858) from femur and tibia-derived BM were stimulated for 3h in StemSpan SFEM II medium (StemCell Technologies) supplemented with L-glutamine (20 mM, Gibco), mSCF, mTPO and hIGF2 (20 ng/ml each) plus hIGF2 and mIL-3 (10 ng/ml each, Peprotech) before nucleofection followed by 20h of culture without mIL-3 prior to harvest for CAST-Seq.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was isolated from cells using theNucleoSpin Tissue Mini kit for DNA from cells and tissue (Macherey-Nagel) 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 were purified using the QIAquick® PCR purification 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 a Illumina HiSeq4000 device collecting 2x150 bp paired-end reads.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
u3d4 replicate 2
UNC-u3d4_U3_OVL2_FINAL.xlsx
UNC-u3d4_D4_OVL2_FINAL.xlsx
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| Data processing |
library strategy: CAST-seq
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: mm10
Supplementary files format and content: matrix table of translocation sites detected with CAST-Seq pipeline with first gRNA. It includes the genomic coordinates of translocated sites, read coverage, alignment score against gRNA, translocation event classification and nearest genes annotation.
Supplementary files format and content: matrix table of translocation sites detected with CAST-Seq pipeline with second gRNA. 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 |
Feb 14, 2023 |
| Last update date |
Jul 04, 2023 |
| Contact name |
Geoffroy Andrieux |
| Organization name |
University clinics Freiburg
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| Street address |
Breisacherstr 153
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| City |
Freiburg |
| ZIP/Postal code |
79110 |
| Country |
Germany |
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| Platform ID |
GPL21103 |
| Series (2) |
| GSE225269 |
Gene Editing of Hematopoietic Stem Cells Restores T Cell Response in a Familial Hemophagocytic Lymphohistiocytosis Model |
| GSE225541 |
Gene Editing of Hematopoietic Stem Cells Restores T Cell Response in a Familial Hemophagocytic Lymphohistiocytosis Model |
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| Relations |
| BioSample |
SAMN33285343 |
| SRA |
SRX19364161 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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