|
| Status |
Public on Apr 03, 2024 |
| Title |
P18_alt_3 |
| Sample type |
SRA |
| |
|
| Source name |
Blood
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Blood
cell line: GM24385
cell type: B-Lymphocyte
genotype: WT
treatment: Nucleofection of Cas9 and gRNA targeting the REF or ALT alelle
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The gDNA of lymphoblastoid cells nucleofected with RNPs was extracted 3 days after CRISPR delivery using the Qiagen Blood and Tissue Kit (Qiagen 69506) according to the manufacturer’s instructions.
Approximately 100ng of gDNA from each sample was used for locus amplification using the primers listed in Supplementary Table 8. Amplicon libraries were generated as described elsewhere 65 with the following modifications: a first round of amplification using the NEBNext UltraII Q5 Master Mix (M0544) was performed with 33 cycles. The amplified DNA was purified using a 1× volume of DNA AMPure XP beads (Beckman Coulter A63987) and the entire purified product was used for a second round of PCR with primers containing p5 and p7 sequences for Illumina sequencing (Supplementary Table 8). Amplicons were pooled and sequenced in a MiniSeq sequencer with the MiniSeq Mid Output Kit (Illumina FC-420-1004) in single-read mode and 150 cycles.
Amplicon-seq
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
CRISPResso version 2.2.12 called with following parameters: --amplicon_min_alignment_score 50 --quantification_window_size 10 --quantification_window_center -3 --exclude_bp_from_left 0 --exclude_bp_from_right 0 --ignore_substitutions --plot_window_size 20 --min_frequency_alleles_around_cut_to_plot 0
Assembly: UCSC Human Genome version hg38 (timestamp 2014-01-15 21:14) downloaded from http://hgdownload.soe.ucsc.edu/goldenPath/hg38/bigZips/hg38.fa.gz
Supplementary files format and content: TAR of tab-separated files showing the number of modifications for positions in the quantification window of the amplicon. The first row shows the amplicon sequence in the quantification window, and successive rows show the number of reads with insertions (row 2), insertions_left (row 3), deletions (row 4), substitutions (row 5) and the sum of all modifications (row 6). Additionally, the last row shows the number of reads aligned.
|
| |
|
| Submission date |
Nov 17, 2023 |
| Last update date |
Apr 03, 2024 |
| Contact name |
Sergi Sayols |
| Organization name |
Institute of Molecular Biology, Mainz
|
| Street address |
Ackermannweg 4
|
| City |
Mainz |
| ZIP/Postal code |
55128 |
| Country |
Germany |
| |
|
| Platform ID |
GPL18573 |
| Series (2) |
| GSE223772 |
Linking CRISPR/Cas9 double-strand break profiles to gene editing precision with BreakTag |
| GSE227604 |
Linking CRISPR/Cas9 double-strand break profiles to gene editing precision with BreakTag [indels] |
|
| Relations |
| BioSample |
SAMN38303495 |
| SRA |
SRX22561191 |
