|
| Status |
Public on Sep 15, 2022 |
| Title |
CH12_LAM-HTGTS_WT_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
CH12
|
| Organism |
Mus musculus |
| Characteristics |
cell line: CH12
genotype: WT
|
| Treatment protocol |
Cells were infected with pMXAIDER vector and electroporateed with Cas9-sgRNA-Myc/Cas9-sgRNA-Irf2bp2
|
| Growth protocol |
CH12 cells cultured in complete RPMI medium wit IL4/CD40/TGF beta stimulation.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted and c-Myc/Irf2bp2 genomic region was biotin isolated and PCR enriched
Libraries were prepared following the nanopore library preparation protocol Ligation Sequencing Kit SQK-LSK109
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
MinION |
| |
|
| Description |
LAM-HTGTS
|
| Data processing |
Library strategy: LAM-HTGTS
LAM-HTGTS
Base-calling and demultiplexing:
Raw nanopore fast5 files were basecalled using the Guppy basecaller from Oxford Nanopore Technologies (ONT) 3.6.0+98ff765.
Resulting reads were demultiplexed by detecting barcodes with a custom fork of qcat (https://github.com/nanoporetech/qcat) and splitting reads into chunks at every detected barcode site, assigning the resulting subreads to the majority vote of the detected barcode set.
Only reads >= 50 bp were used to remove linker-linker pairs.
Alignment and downsampling:
Reads were aligned using bwa mem v0.7.17-r1188 and primary alignments were retained with samtools -F 0x0100.
Reads mapping to the bait regions (Myc chr15:61794274-61844667; Irf2bp2 chr8:129089061-129140470) were extracted.
Downsampling factors for matched replicates between conditions were calculated and downsampling was performed using picardTools MarkDuplicates (https://broadinstitute.github.io/picard/) v2.18.27.
Translocation and hotspot calling:
The pysam (https://github.com/pysam-developers/pysam) module was used to extract reads from the bait regions and translocation breakpoints were extracted from all supplementary alignments in the SA bam tag outside the bait region.
Translocations also appearing the respective control samples were subtracted for the final translocation sets.
Hotspots on the resulting translocation bed files were called using hot_scan (Version from October 17, 2013) with window width 10kb, significance level 0.05 and unadjusted p-values.
Genome_build: NCBI mm9
Supplementary_files_format_and_content: Tab-delimited tables with translocation quantifications and log2-foldchanges on confident hotspot sets.
|
| |
|
| Submission date |
Nov 19, 2020 |
| Last update date |
Sep 15, 2022 |
| Contact name |
Tobias Neumann |
| Organization name |
IMP
|
| Street address |
Campus-Vienna-Biocenter 1
|
| City |
Vienna |
| ZIP/Postal code |
1030 |
| Country |
Austria |
| |
|
| Platform ID |
GPL24973 |
| Series (2) |
| GSE161818 |
DNA replication timing directly regulates the frequency of oncogenic chromosomal translocations |
| GSE161822 |
DNA replication timing directly regulates the frequency of oncogenic chromosomal translocations |
|
| Relations |
| BioSample |
SAMN16837321 |
| SRA |
SRX9532360 |
