|
| Status |
Public on Jul 25, 2023 |
| Title |
RL379_2016_11_06_R1p36_150716_Day4 |
| Sample type |
SRA |
| |
|
| Source name |
mESC neural diff 4 days induction
|
| Organism |
Mus musculus |
| Characteristics |
cell line: R1
cell type: mESC neural diff 4 days induction
cell culture: in vitro
age: 4 days
|
| Treatment protocol |
High potassium-mediated depolarisation was performed by washing cells with low K+ buffer, transferring to high K+ buffer for 5 min, washed again in low K+ buffer and either harvested directly for RNA, immediately fixed for ICC, or chased in growth medium for the appropriate times prior to fixation or RNA extraction.
|
| Growth protocol |
mESC cells lines R1 and G4 were maintained on gamma-irradiated mouse embryonic fibroblasts in mESC medium with LIF. Cells were fed daily and split based on confluency. Neural differentiation was initiated by dissociating the mESC colonies using Accutase and excess feeder cells were removed from the cell suspensions by panning on gelatin-coated plates. Dissociated cells were counted and transferred to ultra-low attachment cell culture plates at a dilution of 220,000 cells/ml, in differentiation medium. Neural induction was continued for 8 days, medium was changed every two days, and supplemented with 5 µM retinoic acid for the final 4 days. Cell aggregates were dissociated using Accutase, single cell selected through a 40 µm cell strainer, and plated onto poly-ornithine / laminin-coated plates in N2 medium. After 2 days, cells were changed into N2B27 which was changed every 4 days for 12 days, following which 50% media changes were performed for the remaining culture time.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Quiagen Blood and Tissue DNA Extraction kit with extended Pretinase K and RNAsA treatment steps.
Lucigen Nxseq Ampfree Low DNA Library Kit and Zymo EZ DNA Methylation Direct Kit for MethylC libraries.
|
| |
|
| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 1500 |
| |
|
| Description |
ESC-derived wild type neural differentiation
|
| Data processing |
adapter trimming using Methpipe pipeline as described in Lister et al Science 2013
mapping reads to mm10 genome using Bowtie as described in Lister et al Science 2013
merging mapped runs, convert to .slam, sort, collapse, trim reads using Methpipe pipeline as described in Lister et al Science 2013
stack creation and hammer correction using Methpipe pipeline as described in Lister et al Science 2013
make allC tables and count global statistics using Methpipe pipeline as described in Lister et al Science 2013
convert stacks to bedgraph files using simple awk script
generate bigwigs using bedgraphtobigwig v4
Genome_build: mm10
Supplementary_files_format_and_content: stacks.txt files containing all mapped methylated and total cytosines, bigWigs for coverage containing all mapped cytosines, bigWigs for mC containing percentage of methylated cytosines for contexts CG and CH
|
| |
|
| Submission date |
Sep 09, 2019 |
| Last update date |
Jul 25, 2023 |
| Contact name |
Ryan Lister |
| E-mail(s) |
ryanlister@gmail.com
|
| Phone |
61864884407
|
| Organization name |
The University of Western Australia
|
| Street address |
35 Stirling Highway
|
| City |
Perth |
| State/province |
WA |
| ZIP/Postal code |
6009 |
| Country |
Australia |
| |
|
| Platform ID |
GPL18480 |
| Series (1) |
| GSE137098 |
Embryonic stem cell-derived neurons as a model system for epigenome maturation during development |
|
| Relations |
| BioSample |
SAMN12715452 |
| SRA |
SRX6818814 |
