 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jun 01, 2024 |
| Title |
WT_NPC_WGBS_Rep2 |
| Sample type |
SRA |
| |
|
| Source name |
Neural progenitor cells
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Neural progenitor cells from ganglionic eminence
strain: C57BL/6
genotype: Wildtype
age: Embryonic day 15.5
fetus id: 2982-2WT
|
| Growth protocol |
Ganglionic eminences of fetal brains were harvested, dissociated with Accutase solution (Sigma A6964), and cultured in ultra-low attachment plates (Corning 3471) with advanced DMEM/F-12 (Gibco 12634-010) supplemented with N-2 (Gibco 17502048), B-27 (Gibco 12587010), GlutaMAX (Gibco 35050061), P/S (Sigma V900929), heparin (5 ug/ml), EGF (20 ng/ml) and FGF (20 ng/ml). Fresh medium was added every 3 days, and neurospheres were dissociated with Accutase and passed every 5 days.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
2 million cultured cells were harvested, washed in PBS and lysed with NP40 lysis buffer (0.32 M sucrose, 5 mM CaCl2, 3 mM Mg(Ace)2, 0.1 mM EDTA, 10 mM Tris pH8, 0.1% NP40). Nuclei were collected with centrifugation, incubated with 5 μl of 10 mg/ml RNase A (Sigma R6513) in 500 μl PK lysis buffer (1 M Tris pH8, 50 mM EDTA, 2% SDS, 2 M NaCl) at 37 ℃ for 15 minutes, and then with 10 μl of 20 mg/ml proteinase K (Sigma P2308) at 52 ℃ overnight with rotation. The samples were then added with phenol/chloroform (1:1) (Solarbio P1021), mixed with vortex, and centrifugated with 15000 g at RT for 10 minutes. The supernatant was taken and precipitated with isopropanol. The precipitated genomic DNA (gDNA) was washed in 70% ethanol, air dried and dissolved in buffer EB (Qiagen 19086).
The gDNA was spiked in with 1% of unmethylated lambda DNA (Promega D1521) for efficiency validation of bisulfite conversion. 500 ng of gDNA was taken and sonicated to ~250 bp with Covaris M220 ultrasonicator. The DNA fragments were end-repaired and A-tailed with NEBNext Ultra II End Repair/dA-Tailing Module (NEB E7546), and then ligated with NEBNext Multiplex Oligos for Illumina (Methylated Adaptor, Index Primers Set 1, NEB E7535). After size-selected to 300~400 bp with Ampure XP beads (Beckman A63881), DNA was bisulfite converted with EZ DNA methylation kit (Zymo Research D5001), PCR amplified with KAPA HiFi HotStart Uracil+ ReadyMix (Kapa Biosystems KK2801), and the library was finally size-selected to 300~500 bp with Ampure XP beads. After analysis with Agilent Tapestation 4200, the library was sent for deep sequencing on Illumina NovaSeq 6000 (paired-end, 150bp).
|
| |
|
| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
WGBS reads were filtered and trimmed with “fastp” (version 0.23.4, -q 20 -u 50 -n 5).
Quality control was conducted by FastQC (version 0.11.9).
High quality clean reads were aligned to mouse genome mm10 and lambda phage genome with Bowtie2 (version 2.2.3) in default settings.
Aligned reads were deduplicated with “deduplicate_bismark” function of Bismark (version 0.22.1).
Methylation measurement was extracted with “bismark_methylation_extractor” function of Bismark (--no_overlap --ignore_r2 2 --comprehensive), and the bisulfite conversion rate was calculated according to the methylation level of spiked-in lambda DNA.
Assembly: mm10
Supplementary files format and content: COV: Coverage text files, tab delimited
|
| |
|
| Submission date |
Nov 13, 2023 |
| Last update date |
Jun 01, 2024 |
| Contact name |
Yan Jiang |
| E-mail(s) |
yan_jiang@fudan.edu.cn
|
| Organization name |
Fudan University
|
| Department |
Institutes of Brain Science
|
| Street address |
131 Dongan rd
|
| City |
Shanghai |
| ZIP/Postal code |
200032 |
| Country |
China |
| |
|
| Platform ID |
GPL24247 |
| Series (2) |
| GSE247619 |
Histone methyltransferase SETDB1 silenced SINE B2 retrotransposons and maintained mitotic cell cycle of neural progenitor cells (WGBS) |
| GSE247620 |
Histone methyltransferase SETDB1 silenced SINE B2 retrotransposons and maintained mitotic cell cycle of neural progenitor cells |
|
| Relations |
| BioSample |
SAMN38228958 |
| SRA |
SRX22506459 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7898086_WT_NPC_WGBS_Rep2.deduplicated.bismark.cov.gz |
171.3 Mb |
(ftp)(http) |
COV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
