|
| Status |
Public on Nov 08, 2021 |
| Title |
80-15-5_MOUSE22_BM |
| Sample type |
SRA |
| |
|
| Source name |
80-15-5_MOUSE22_BM
|
| Organism |
Mus musculus |
| Characteristics |
tissue: BM
mutation: 80-15-5
mouse: MOUSE22
timepoint: Disease
|
| Treatment protocol |
Cells were transduced with SPLINTR barcode libraries in polybrene (8.5 mg/mL) via spinfection (90 minutes at 1250 g).
|
| Growth protocol |
c-Kit positive hematopoietic progenitor cells were cultured in RPMI-1640 medium supplemented with mouse IL-3 (10 ng/mL), human IL-6 (10 ng/mL), mouse SCF (50 ng/mL) 20% fetal bovine serum (FBS), streptomycin (100 ug/mL), penicillin (100 units/mL) and 2 mM glutamax in 5% CO2 at 37 °C.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Viable cells were sorted on the BD FACSAria III directly into Eppendorf tubes. The cells were then washed twice with PBS + 0.04% BSA.
Cells were lysed in 40 uL Viagen lysis buffer containing 0.5 mg/mL Proteinase K (Invitrogen) and split into technical replicates. Barcodes in cell lysate were then amplified following two rounds of PCR using Q5 polymerase (NEB). The first PCR amplified barcode specific DNA and added partial Illumina sequencing primer binding sites. PCR primers containing staggers to account for low sequence diversity during sequencing. In the second round PCR, the sequencing primer binding sites were completed, P5 and P7 flow cell regions were added and samples received a well-specific 8bp dual index (96 different indices in total).
DNA-Seq
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
KF09_COMBO-80-15-5_filtered_collapsed_CPM.txt
|
| Data processing |
Barcode sequences were extracted from fastq files based on the known constant sequences flanking the random barcodes and subsequently trimmed to remove these regions such that only the random barcode sequences remained.
Barcode containing reads were further filtered to keep only those barcodes that (a) showed a correct upstream constant region (5’- CGATTGACTA-3’), (b) showed the expected barcode pattern (c) did not contain N residues, and (d) possessed an average phred quality of 30 or higher across the length of the barcode.
Barcode reads were then mapped to the respective SPLINTR reference library using bowtie v1.2.2 allowing up to 2 mismatches.
Barcode count data were imported into R v3.6 for further analysis.
Genome_build: SPLINTR reference library
|
| |
|
| Submission date |
Nov 17, 2020 |
| Last update date |
Nov 08, 2021 |
| Contact name |
Mark Dawson |
| E-mail(s) |
mark.dawson@petermac.org
|
| Organization name |
Peter MacCallum Cancer Centre
|
| Street address |
305 Grattan Street
|
| City |
Melbourne |
| State/province |
VIC |
| ZIP/Postal code |
3000 |
| Country |
Australia |
| |
|
| Platform ID |
GPL19057 |
| Series (2) |
| GSE161672 |
Identifying non-genetic determinants of malignant clonal fitness at single cell resolution (clonal competition barcode-seq) |
| GSE161676 |
Identifying non-genetic determinants of malignant clonal fitness at single cell resolution |
|
| Relations |
| BioSample |
SAMN16822786 |
| SRA |
SRX9522941 |
