 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jun 30, 2023 |
| Title |
BS Immortalised MEF cells, subclonal population 1-2 |
| Sample type |
SRA |
| |
|
| Source name |
MEF1-2
|
| Organism |
Mus musculus |
| Characteristics |
cell line: MEF1-2
cell type: Immortalised mouse embryonic fibroblasts (MEFs)
|
| Growth protocol |
Mouse embryonic fibroblasts (MEFs) were established from E13.5 C57BL/6J mouse embryos. MEFs were grown at 37°C and 5% CO2 in high glucose DMEM GlutaMAX™ (ThermoFisher, cat no. 31966021) supplemented with 10% FBS (Gibco, 11550356) and 1% penicillin/streptomycin solution and passaged with trypsin/EDTA. MEFs were immortalised by serial passaging the cells through crisis phase. Isolation of single immortalised MEF cells was performed by flow cytometry (MoFlo Astrios Cell Sorter) to 96-well plates. The isolated single cells were grown into clonal cell lines by successive passaging from 96-well, 48-well, 24-well, 12-well, and 6-well plates to 10cm dishes where they were grown to 75% confluency to yield 1-2 million cells per clonal line for DNA/RNA extraction. The parental lines, from which the clonal lines were derived, were also grown to 75% confluency on 10cm dishes to yield 1-2 million cells for DNA/RNA extraction.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA extraction was performed with the Qiagen AllPrep DNA/RNA Mini Kit (cat no. 80204).
Target capture bisulfite-sequencing (tcBS-seq) libraries were generated using the SureSelectXT Methyl-seq Library Preparation Kit with the following specifications. 1µg of DNA was sonicated to an average size of 200bp with the Covaris E220 (Duty Factor = 30%, PIP = 100, Cycles per Burst = 1000, Treatment Time = 95, Bath Temperature = 7°C, with intensifier fitted) using 50µl microTUBEs (cat no. 520166) and 24-place rack (cat no. 500308). Following end repair, dA tailing, and adaptor ligation, libraries were hybridised to single-stranded RNA probes homologous to 297,000 regions in the mouse genome (SureSelectXT Methyl-Seq Capture Probes, cat no. 931052). After purification, the libraries were bisulfite converted using Zymo Research’s Methylation-Gold Kit (cat no. D5005) and PCR amplified for 8 cycles. The PCR amplified bisulfite-treated libraries were then purified, indexed by PCR amplification (6 cycles), and purified again. All purification steps took place using Ampure XP beads (Beckman Coulter, cat no. A63881). The final libraries were then quality checked and quantified for multiplexing using the Bioanalyzer High-Sensitivity DNA kit (Agilent, 5067-4626) and Qubit dsDNA HS Assay kit (Thermo Scientific, Q32854). The multiplexed libraries were sequenced as 150 bp paired-end reads on the Illumina Novaseq 6000 SP.
|
| |
|
| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
tcBS-seq reads were trimmed by Trim Galore (v0.6.0).
tcBS-seq reads were aligned to mm10 using Bismark.
tcBS-seq reads with map quality score less than 10 were excluded and were further filtered by M-bias: for MEF-1 and MEF-2 data, we excluded the first two bp on both paired reads and the last bp on Read 2.
tcBS-seq read data was extracted using bismark_methylation_extractor and analysed in R (v3.6.1) to combine data from all cell lines and compare with genomic annotations and other datasets.
For the all_clonal_MEF_methylation_data processed file, we used read coverage across the individual tcBS-seq libraries to look for enrichment of reads on individual chromosomes to determine coverage-based karyotypes for our immortalised MEF cell lines and find that chromosomes 12, 18, and 19 exhibit aneuploidy in at least one of the cell lines. For subsequent analyses, we removed the CpGs on the aneuploid chromosomes, as well as those on the X chromosome. Additionally, we thresholded the data for CpGs with greater than or equal to 10x coverage in all 16 sequencing libraries, retaining ~1.2 million CpGs (or ~5% of CpGs in the mouse genome) with a median coverage of 32 reads per CpG per tcBS-seq dataset.
Assembly: mm10
Supplementary files format and content: CpG_combine* files are tab delimited with the following four columns: chromosome, genomic locus (bp), unmethylated read counts, methylated read counts
Supplementary files format and content: all_clonal_MEF_methylation_data is a tab delimited file with the following 12 columns: chromosome, start locus of CpG, end locus of CpG, read coverage, unmethylated read count, methylated read count, percentage of methylation, cell line, cell type, fidelity score, neighbour similarity score, cpg density
|
| |
|
| Submission date |
Jun 02, 2023 |
| Last update date |
Jun 30, 2023 |
| Contact name |
Anne Ferguson-Smith |
| E-mail(s) |
afsmith@gen.cam.ac.uk
|
| Organization name |
Cambridge University
|
| Department |
Genetics
|
| Street address |
20 Downing Place
|
| City |
Cambridge |
| ZIP/Postal code |
CB2 3EJ |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL24247 |
| Series (2) |
| GSE234029 |
Target capture bisulfite sequencing (tcBS-seq) to evaluate clonal methylation inheritance [BiSulfite-seq 2] |
| GSE234695 |
Target capture bisulfite sequencing (tcBS-seq) to evaluate clonal methylation inheritance |
|
| Relations |
| BioSample |
SAMN35571745 |
| SRA |
SRX20579388 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7441385_CpG_combine_SLX-19089.SXTC01.HTFMWDRXX.s_2.r_1_val_1_bismark_bt2_pe.mqual_ge10.txt.gz |
20.1 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
|
|
|
|
 |
