 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Feb 13, 2020 |
| Title |
E14 DNMT1 KO single cells- Library 3 |
| Sample type |
SRA |
| |
|
| Source name |
Embryonic stem cells
|
| Organism |
Mus musculus |
| Characteristics |
cell line: E14tg2a
strain: B6
|
| Treatment protocol |
Six gRNA sequences targeting three exons of mouse Dnmt1 were used. Phosphorylated BbsI compatible restriction overhangs were added to gRNA top and bottom oligos and resuspended at 100 M in nuclease-free water. Annealing of the oligos was performed in 1x ligation buffer (NEB) using the following program: 97°C for 5 minutes, ramp down by 1°C per 1 minute to 20°C. The pX330 CRISPR-Cas9-GFP gRNA plasmid was mixed with 0.1 M gRNA oligo. The reaction was simultaneously digested with BbsI (NEB) and ligated with T4 DNA ligase (NEB) overnight at 16°C. Ligation reactions were transformed into DH5 competent cells and subsequently sequenced using Sanger dideoxy sequencing to confirm the correct insert. All six pX300-gRNA plasmids were pooled and 1 g was transfected into 2 million E14tg2a cells using Lipofectamine (Life Technologies). A separate pX300 empty vector was also transfected into E14tg2a to serve as a negative control. Two days later, single GFP positive cells were sorted into 384-well plates (BioRad) and subjected to scMspJI-seq.
|
| Growth protocol |
Cells were grown on 0.1% gelatin in ES cell culture media; DMEM (1×) high glucose + glutamax (Gibco), supplemented with 10% FCS (Greiner) 100 µM β-mercaptoethanol (Sigma), 100 µM Non-essential amino acids (Gibco), 50 µg/mL Pen/Strep (Gibco) and 1000 U/mL ESGRO mLIF (Millipore).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were isolated using hyaluronic acid (Sigma) and trypsin (Life Technologies) and sorted into 384-well plates containing lysis buffer and Vapor-lock.
Single cells were incubated at 50C for 15 hours, 75C for 20 minutes and 80C for 5 minutes. Glucosylation of 5hmC sites followed by dispensing 0.1 L of T4-BGT (NEB), 0.1 L of UDP-Glucose (NEB), 0.05 L of 10X NEB Buffer 4 and 0.25 L of nuclease-free water (final vol 0.5mL). After incubation at 37C for 16 hours, 0.5 L of the following reaction mixture is added: 0.1L of 25g/L Qiagen Protease, 0.05L of 10X NEB Buffer 4 and 0.35 L of nuclease-free water. The plate is then incubated at 50 o C for 5 hours, 75 o C for 20 minutes and 80 o C for 5 minutes. Thereafter, gDNA is digested by the restriction enzyme MspJI by the addition of 0.5 L of the following reaction mixture: 0.02 L of MspJI (NEB), 0.12 L of 30X enzyme activator solution (NEB), 0.05 L of 10X NEB Buffer 4 and 0.31 L of nuclease-free water. The digestion is performed at 37 o C for 5 hours followed by heat inactivation of MspJI at 65 o C for 20 minutes. Next, 0.2 L of cell-specific double-stranded adaptors are added to individual wells and these adapters are ligated to the fragmented gDNA molecules by adding 0.8 L of the following reaction mixture: 0.07 L of T4 DNA ligase (NEB), 0.1 L of T4 DNA ligase buffer (NEB), 0.3 L of 10 mM ATP (NEB) and 0.33 L of nuclease-free water. The ligation is performed at 16 o C for 16 hours. Next, wells containing unique cell-specific adapters are pooled using a multichannel pipette and incubated with 0.8X Agencourt Ampure (Beckman Coulter) beads for 30 minutes, washed twice with 80% ethanol and resuspended in 6.4 L of nuclease-free water. In vitro transcription and Illumina library preparation is performed as described previously in the scAba-seq protocol.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Library strategy: scMspJ1-seq
Fastq files are parsed for library barcodes. Read 1 was mapped to the mm10 or hg19 build using the Bowtie2, filtered for unique mapping full-length reads and demultiplexed. 5mCG positions are obtained from the sequencing data using custom scripts in R and Perl identifying CG dinucleotides in the mm10 or hg19 genome at the expected distance from the mapped read position. Finally, all PCR duplicates are removed. Computer codes will be made available upon request.
Hairpin bisulfite sequencing was performed on bulk mouse embryos samples ( 2-cell to 64 cell stage). The bulk embryos were treated with protease treatment (1 uL of 25 ug/uL Qiagen Protease, 1 uL of 10X NEB Buffer 4, and 8 uL of nuclease free water). Then, 0.5 ng of genomic DNA was digested with 20 mL of MspI master mix (1 uL of MspI R0104L, 2 mL 10X NEB Cutsmart buffer in a total volume of 20 uL) and incubated at 37 C for an hour. After digestion, the digested genomic DNA was ligated with 1 uL of 10 uM phosphorylated hairpin oligo and ligation master mix (1 mL of NEB T4 ligase M0202M, 1 uL of 10X NEB T4 Ligase buffer, 2 uL of 10mM ATP, 5 uL of nuclease free water) and incubated overnight at 16 C. For purification of the ligation mixture, we used Dynabeads? M-280 Streptavidin and followed the recommended protocol with the following exceptions: the bead-ligation mixture was incubated for at least an hour at RT on a rotator and cold 10 mM Tris-HCl wash step was included. Subsequently, we performed BS-seq on the sample using the protocol Clark, et al. 2017. After sequencing the libraries on a Miseq 300x300 or NextSeq 75x75 pair-end kit, we used HBS-tools (Sun, MA et al. 2015) and custom perl scripts were used to analyze the methylated CpG dyads. Hairpin oligo (G/iMe-dC/iMe-dC/G/iMe-dC/iMe-dC/GG/iMe-dC/GG/iMe-dC/AAG/iBiodT/GAAG/iMe-dC/iMe-dC/G/iMe-dC/iMe-dC/GG/iMe-dC/G) was resuspended in 100 uM of Low-TE. The hairpin oligo was phosphorylated (1 uL of 100 uM hairpin oligo, 3 uL of 10X T4 Ligase Buffer, 1 uL T4 PNK and 5 uL of nuclease free water) and incubated at 37 C for an hour. Subsequently the phosphorylated oligo heated at 94 C and placed in ice water to generate the loop.
Published scNMT libraries (GSE109262) analyzed for the bisulfite results were processed as described previously. The first nine bases of the raw reads were trimmed using Trim Galore (v0.5.0) and mapped using Bismark to the mouse genome (mm10) with the 129/CAST background. SNPs specific to 129/CAST mouse genome were prepared using SNPsplit (v0.3.2) and a list of known variant call files from the Mouse Genomes Project (http://www.sanger.ac.uk/resources/mouse/genomes/). After mapping with Bismark, duplicate sequences were removed and CpG methylation calls were extracted with strand-specific information. Further data analysis and visualization of the methylation calls used custom scripts that will be made available upon request.
Genome_build: mm10, hg19
Supplementary_files_format_and_content: mText files with 4 columns: Single Cell ID, chromosome, coordinate, strand, CNNR motif and strain [mouse only])
|
| |
|
| Submission date |
Nov 05, 2019 |
| Last update date |
Feb 13, 2020 |
| Contact name |
Maya Sen |
| E-mail(s) |
m.sen.87@gmail.com
|
| Organization name |
Hubrecht Institute
|
| Street address |
8 Uppsalalaan
|
| City |
Utrecht |
| ZIP/Postal code |
3584CT |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL19057 |
| Series (1) |
| GSE139984 |
Strand-specific single-cell methylomics reveals distinct modes of DNA demethylation dynamics in preimplantation mouse and human embryos |
|
| Relations |
| BioSample |
SAMN13218858 |
| SRA |
SRX7101512 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4151087_D3DNMT3_5mCReads_se_half5mc_correctpos_withstrand_stringent_simplified_sort_rmdup.txt.gz |
14.8 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
