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| Status |
Public on Dec 26, 2018 |
| Title |
181102_snm3C_B4_AD001_indexed |
| Sample type |
SRA |
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| Source name |
Mouse Embryonic Stem Cell Line E14
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| Organism |
Mus musculus |
| Characteristics |
Sex: Male
genome build: mm10
library strategy: sn-m3C
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| Growth protocol |
Mouse ES cells (E14TG2a) were purchased from American Type Culture Collection (ATCC CRL-1821). ES cells were grown in DMEM media (Corning 10-013-CV) supplemented with 10% HyClone FBS (Fisher SH3007003E), 1X MEM Non-essential amino acids (ThermoFisher 11140050), 1X Glutamax supplement (ThermoFisher 35050061), 1X ß-mercaptoethanol (Millipore ES-007-E), 100U/mL Penicillin-Streptomycin (ThermoFisher 15140122), and 1000U/mL Leukemia Inhibitory Factor (Millipore ESG1107). ES cells were cultured in feeder free conditions on 0.5% gelatin coated plates. GM12878 cells were obtained from Coriell Institute for Medical Research. GM12878 cells were grown in RPMI-1640 medium (ThermoFisher 11875093) supplemented with 15% Fetal Bovine Serum (Corning 35-010-CV) and 100U/mL Penicillin-Streptomycin (ThermoFisher 15140122). NMuMg cells (RBRC-RCB2868) were obtained from the RIKEN BioResource Center. NMuMg cells were grown in DMEM (Corning 10-013-CV) with 10% Fetal Bovine Serum (Corning 35-010-CV), 10ug/mL Insulin (ThermoFisher 12585014), and 100U/mL Penicillin-Streptomycin (ThermoFisher 15140122). All cell lines were routinely tested for mycoplasma contamination and tested negative.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
in situ Hi-C was performed as previously described using the MboI restriction enzyme. in situ 3C experiments were performed based on the previously described in situ Hi-C protocol with minor modifications. Briefly, prior to fixation, adherent cells were trypsinized, counted, and collected by centrifugation, and suspension cells were counted and collected by centrifugation. Cells were resuspended in culture media at a concentration of 1x106 cells per mL of media and fixed in 1% formaldehyde for 10 minutes at room temperature with shaking. For standard species mixture experiments, equal numbers of mouse and human cells were combined into a single tube prior to fixation. For the 1:10 dilution species mixture experiment, cells were resuspended at a concentration of 1x105 cells per mL of media prior to fixation. For the species mixture experiments where samples were mixed after fixation, each cell type was fixed independently as described above and combined at later stages in the protocol. in situ Hi-C samples were digested with the MboI restriction enzyme and processed as described previously. For in situ 3C experiments, samples were digested with DpnII enzyme overnight at 37ºC with gentle mixing. The following day, the sample was incubated at 62ºC for 10 minutes to inactivate the restriction enzyme. The typical biotin fill in step in the Hi-C protocol is omitted. The sample is then ligated for 4 hours at room temperature with T4 DNA ligase in the same manner as in in situ Hi-C experiments. The sample is then stained with Hoechst (0.1ug/uL) for the final 30 minutes of the ligation step. The sample is then passed through a 40 uM nylon cell strainer (Corning 431750) into a FACS tube prior to sorting. As a quality control step, 10% of the sample is taken for conventional library preparation and sequenced using shallow sequencing on a MiSeq. Libraries for bulk and single-cell methylomes were generated using snmC-seq2. A detailed step-by-step bench protocol for snmC-seq2 is provided as Supplement Methods in Luo et al. (2018) 23. Bulk methylome libraries were prepared manually using individual tubes. Single-cell methylome libraries were prepared using a Tecan Evo 100 robotic platform as described in Luo et al. (2018) 23. Sequencing libraries were sequenced using Illumina MiSeq and HiSeq 4000 instruments in PE150 mode.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Raw reads were trimmed first 25bp and last 3bp of both read1 and read2 to remove random primer sequence and adaptase low complexity tails. For the alignment, Bismark is used24. C to T converted and G to A converted reference genomes are prepared for mm10 and hg38 using bismark_genome_preparation. Each read end is mapped separately using Bismark with Bowtie1 with read1 as complementary (always G to A converted) and read2 (always C to T converted) as original strand. After first alignment, unmapped reads are retained and splitted into 3 pieces by 40bp, 32bp, and 40bp after removing 5bp of both ends results in having 6 reads (read1 and read2). Six reads derived from unmapped reads are mapped separately using Bismark Bowtie1. All aligned reads are merged into BAM using picard that is query name sorted. The fragments with all the mapped reads aligned to the same positions are considered as duplicates and removed and DNA methylation tracks are generated. For each fragment, the outermost aligned reads are chosen for the chromatin conformation map generation.
Supplementary_files_format_and_content: tab delimited text files of methylcytosine calls; columns in allc files are: column 1 - chromosome; column 2 - position; column 3 - strand; column 4 - class; column 5 - mC reads; column 6 - total reads; column 7 - methylated (Boolean value indicating the result of statistical test for methylated cytosines)
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| Submission date |
Dec 26, 2018 |
| Last update date |
Jan 02, 2019 |
| Contact name |
Joseph R Ecker |
| E-mail(s) |
ecker@salk.edu
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| Phone |
8584534100
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| Organization name |
HHMI-Salk-Institute
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| Department |
Genomic Analysis Laboratory
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| Lab |
Ecker lab
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| Street address |
10010 North Torrey Pines Road
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
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| Platform ID |
GPL21103 |
| Series (1) |
| GSE124391 |
Single-cell multi-omic profiling of chromatin conformation and DNA methylome |
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| Relations |
| BioSample |
SAMN10645587 |
| SRA |
SRX5180859 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3531433_181102_snm3C_B4_AD001_indexed_contacts.txt.gz |
728.9 Kb |
(ftp)(http) |
TXT |
| GSM3531433_allc_181102_snm3C_B4_AD001_indexed.tsv.gz |
30.1 Mb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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