|
| Status |
Public on Sep 11, 2022 |
| Title |
Cyclin B1 -ve [A11] |
| Sample type |
SRA |
| |
|
| Source name |
colorectal tumour
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: colorectal tumour
cell line: COLO205
genotype: BRAF V600E
treatment: selumetinib (48h, 1uM)
|
| Treatment protocol |
COLO205 cells were treated with 1uM selumetinib for 48 hours
|
| Growth protocol |
COLO205 cell lines were provided by the laboratory of Dr. Simon J Cook (Babraham Institute). Cells were cultured in RPMI-1640 media supplemented with 10% (v/v) foetal bovine serum, penicillin (100 U/mL), streptomycin (100 mg/mL) and 2 mM glutamine at 37 °C in a humidified incubator with 5% (v/v) CO2.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were glyoxal-fixed and stained for CCNB1 as previously described (Channathodiyil and Houseley, 2021) and single cells sorted by flow cytometry to collect CCNB1 positive and CCNB1 negative cells in a 96-well plate.
Bisulfite conversion was carried out using EZ Methylation Direct bisulfite reagent (Zymo) and desulfonation and purification of converted DNA with magnetic beads (Zymo) on the Bravo automated workstation (Agilent) with DNA eluted into the master mix for the first-strand synthesis. Random priming and extension were performed followed by purification of the resulting DNA fragments. Second strand synthesis was then performed and the resulting fragments amplified with 14 PCR cycles. Amplified libraries were pooled (2 pools of 48 libraries each) and purified using 0.7× Ampure XP beads.
Single cell bisulfite libraries were then prepared as (CLark et al 2017)
|
| |
|
| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
Bisulfite converted
GEO report for rows A-D CCNB1 -ve.txt
|
| Data processing |
adapter and quality trimming using Trim Galore (v0.6.2)
mapped to GRCh38 using Bismark v0.22.1
reads were quantified in 5 Mb windows spaced every 2.5 Mb across the genome, with windows of aberrantly high and low read count excluded
Log transformed datasets were normalised per probe by subtracting the median for that probe, which converts data to log2-fold change in copy number relative to average (the average copy number for each probe being the genomic copy number)
Enrichment normalisation for the 10th and 90th percentiles was then applied to each dataset normalise differences in total read count and distribution.
Assembly: GRCh38
Supplementary files format and content: Normalised total read counts for the CCNB1 negative and CCNB1 positive datasets are provided as separate files
|
| |
|
| Submission date |
Sep 08, 2022 |
| Last update date |
Sep 12, 2022 |
| Contact name |
Laura Biggins |
| E-mail(s) |
laura.biggins@babraham.ac.uk
|
| Organization name |
The Babraham Institute
|
| Department |
Bioinformatics
|
| Street address |
Babraham Research Campus
|
| City |
Cambridge |
| ZIP/Postal code |
CB22 3AT |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL18573 |
| Series (2) |
| GSE168604 |
Selumetinib |
| GSE212899 |
Epigenetic and genetic change through cell cycling under selumetinib treatment |
|
| Relations |
| BioSample |
SAMN30725345 |
| SRA |
SRX17488070 |
