|
| Status |
Public on Nov 16, 2018 |
| Title |
F123_singlecellMethyl-HiC_naive_Cell_45-47 |
| Sample type |
SRA |
| |
|
| Source name |
F123 embryonic stem cells
|
| Organism |
Mus musculus |
| Characteristics |
cell line: F123
strain: F1 Mus musculus castaneus x S129/SvJae
cell type: mouse ESC line
culture condition: 2i+LIF
|
| Treatment protocol |
About two million cells were cross-linked with 1% formaldehyde for 10 min at room temperature. Reaction was then quenched with 0.2M Glycine.
|
| Growth protocol |
The F1 Mus musculus castaneus × S129/SvJae mouse ESC line (F123) was cultured in KSR medium as described previously. Cells were collected and plated on 0.2% gelatin-coated feeder-free plates for 30mins before harvesting to remove feeder cell contamination.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cell pellets were washed with cold PBS and lysed with lysis buffer to get nuclei pellets. Nucleus were permeabilized with 0.5% SDS. DNA was in situ digested with 100 units of DpnII (NEB) overnight. The ends of restriction fragments were filled with biotinylated nucleotides and in situ ligated. After reversal of crosslinks, ligated DNA was ethanol precipitate and sheared to a length of ∼400 bp by sonication (Covaris). Sonicated products were pulled down with streptavidin beads. Library construction was then performed on beads. After adapter ligation, beads were suspended in 20ul TE buffer and subjected to bisulfite conversion with EZ DNA Methylation-Gold™ Kit (Zymo). Unmethylated lambda DNA was sonicated and ligated with the same adapter for Methyl-HiC sample and then was spiked in at 0.5% before bisulfite conversion. After conversion, streptavidin beads were removed with magnet and the supernatant were purified. Purified bisulfite converted DNA was amplified with Hifi Hotstart Uracil+Ready Mix (KAPA).
Libraries were prepared according to Illumina's standard DNA library protocol, in which genomic DNA was fragmented by sonication (Covaris), end-repaired, dA-tailed and ligated to cytosine methylated Illumina TruSeq adapter.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
Library strategy: Methyl-HiC
Analysis of methylation data: Raw reads were first trimmed as paired-end reads using Trimmomatic with default parameters to remove the adapters and low quality reads. Trimmed reads were aligned to mm9 using Bismark (v12.5). PCR duplications were removed with Picard (http://broadinstitute.github.io/picard/). Cpg methylation level were calculated by Bis-SNP in bissnp_easy_usage.pl with default parameters.
Analysis of Hi-C data: All sequence data were produced using Illumina paired-end sequencing. Each end of the raw reads was mapped separately to the mm9 reference genome using BWA-mem. Filtered reads were then paired and de-duplicated (Picard). Reads that map to the same fragment were further removed. Contact matrices were generated at different resolution using Juicer pipeline with KR normalization and visualized using Juicebox. Loops were then called by HICCUPS.
Methyl-HiC reads mapping by Bhmem: Raw reads were first trimmed as paired-end reads using Trimmomatic with default parameters to remove the adapters and low-quality reads. All C in reference genome is converted to T to make C_to_T reference genome, and all G is converted to A to make G_to_A reference genome in mm9. On paired end reads, C is converted to T in end 1 and G is converted to A in end 2. The converted end 1 and end 2 are joined as single paired fragment when both of them were mapped to C_to_T reference genome or G_to_A reference genome by BWA-MEM. Only unique mapped and passed VendorQualityFilter reads on both ends were joined.
The best paired reads were selected based on the following priorities: (1) mapping quality on both ends are bigger than 0. (2) the sum of mapping quality score is larger. (3) both ends of reads are mapped into the same chromosome. (4) the sum of alignment score is larger. (5) the sum of mismatches is smaller. (6).the sum of matched cigar string length is larger. Only best paired reads were output for the following analysis. Low quality reads (not unique mapped on both ends, PCR duplicate and mapping quality score < 30) were removed for the following downstream analysis. Only bases with quality score more than 5 were included in the downstream methylation analysis. Details were implemented in Bhmem.java
Genome_build: mm9 (MGSCv37)
Supplementary_files_format_and_content: Bed files contain DNA methylation called from MethylHiC and WGBS data.
Supplementary_files_format_and_content: Bedpe files contain HICCUPS loops and Methylation Concordance called from MethylHiC data.
Supplementary_files_format_and_content: Hic file contains binary contact matrix for in situ HiC data.
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| |
|
| Submission date |
Aug 28, 2018 |
| Last update date |
Nov 16, 2018 |
| Contact name |
Guoqiang Li |
| E-mail(s) |
liguoqiang.jason@gmail.com
|
| Phone |
+86-10-62757927
|
| Organization name |
Biomedical Pioneering Innovation Center,Peking University
|
| Street address |
No.5 Yiheyuan Rd
|
| City |
Beijing |
| State/province |
Beijing |
| ZIP/Postal code |
100871 |
| Country |
China |
| |
|
| Platform ID |
GPL21103 |
| Series (1) |
| GSE119171 |
Long-range epigenetic concordance revealed by simultaneous profiling of DNA methylation and chromosomal conformation changes in bulk and single cell Methyl-HiC |
|
| Relations |
| BioSample |
SAMN09930051 |
| SRA |
SRX4626297 |
