|
| Status |
Public on Apr 03, 2024 |
| Title |
M_UM_REF_pos17 |
| Sample type |
SRA |
| |
|
| Source name |
Blood
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Blood
cell line: GM12878
cell type: B-Lymphocyte
genotype: WT
treatment: digested with HiPlex pool targeting REF alelle
biomaterial provider: https://www.coriell.org/0/Sections/Search/Sample_Detail.aspx?Ref=GM12878&Product=CC
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
gDNA was obtained from Coriell
DNA DSB ends of nuclease-digested gDNA were repaired and 3’ adenylated using the NEBNext® Ultra™ II End Repair/dA-Tailing Module (NEB E7546) as described by the manufacturer with the following modification: Reaction volume was halved by adding half volume of the reagents. Labeling of DSB ends with BreakTag linker ligation was performed using the NEBNext® Ultra™ II Ligation Module (NEB E7595) according to manufacturer’s recommendation with the following modifications: Reaction volume was halved by adding half volume of reagents, and USER enzyme digestion step was omitted. BreakTag linker was used at a final concentration of 50 nM per sample. Labeled DNA was size-selected using DNA AMpure XP beads (0.7x volumes) in order to remove unligated linkers and eluted in nuclease-free H2O. Tagmentation with in-house Tn5 was performed in 10 mM Tris-HCl pH 7.5, 10 mM MgCl2, and 25% dimethylformamide (DMF, Sigma Aldrich 227056). Tagmentation reactions were assembled using 100-200 ng of DNA as input. Hyperactive Tn5 was used at a final concentration of 1.25 ng/µL per reaction. The tagmentation mix was then incubated at 55ᵒC for 5 minutes in a preheated thermocycler followed by termination with 0.2% SDS at room temperature for 5 minutes. Libraries were amplified with the NEBNext® Ultra™ II Q5® Master Mix (M0544). Amplified and barcoded samples were size-selected in order to remove PCR byproducts by performing two consecutive 0.5x volume right-tail + 0.35x volume left-tail size selection using DNA AMpure XP beads. Final libraries were quantified using Qubit dsDNA High Sensitivity Assay kit and fragment size distribution was assessed in a BioAnalyzer High Sensitivity DNA chip
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
library strategy: BreakTag
NGSpipe2go Breaktag pipeline (https://gitlab.rlp.net/imbforge/NGSpipe2go/-/tree/master/pipelines/breaktag)
Reads without breaktag adaptor were filtered out
Reads with breaktag adaptor were mapped using BWA v0.7.15 with “bwa mem” on genome assembly hg38 and filtered out reads with mapping quality below 60
Deduplication of mapped reads based on unique molecular identifiers (UMIs)
Mapped read ends are summed up and reported in BED format
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: Breaktag signal as a proxy of double strand breaks counts, a a standard 6-columns BED containing the “chromosome | start | end | name | breaktag_signal | strand”
|
| |
|
| Submission date |
Jan 26, 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) |
| GSE223769 |
Linking CRISPR/Cas9 double-strand break profiles to gene editing precision with BreakTag [hiplex3] |
| GSE223772 |
Linking CRISPR/Cas9 double-strand break profiles to gene editing precision with BreakTag |
|
| Relations |
| BioSample |
SAMN32927632 |
| SRA |
SRX19187916 |
