|
| Status |
Public on Aug 21, 2018 |
| Title |
KO1_H3K27me3_PE |
| Sample type |
SRA |
| |
|
| Source name |
Hippocampal primary neurons
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Hippocampal primary neurons
development stage: E16.5
days in vitro: 14
strain: 129Sv/Ev
chip antibody: H3K27me3 (Diagenode, pAb-069-050)
|
| Growth protocol |
Mouse hippocampi from embryos 16.5 days post coitum (E16.5) were dissected in cold HBSS and disassociated by adding warm 0.25 % trypsin. One tenth of the trypsin volume of FBS was added to neutralize trypsin. Cells were collected by centrifugation at 200 RCF for 5 minutes and were resuspended and then cultured in Neurobasal Medium (NBM) with 1 % FBS, 1 % P/S, and 1x B27 supplement (all from Invitrogen). Seeding density is 1000 cells/mm2. Primary cells were cultured at 37°C with 5 % CO2 for 14 days before collected.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody.
Libraries were constructed according to NEBNext ChIP-Seq Library Prep Reagent Set for Illumina (NEB). Library fragments between 175 – 225 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Description |
PE and SE raw data was pooled to generate processed data
|
| Data processing |
Basecalls performed using CASAVA (version 1.8.2).
Fastq files were filtered using the following specifications: Minimum Quality:20 and Maximum number of bases allowed outside of quality range: half of the read length.
ChIP-seq reads were aligned to the mm9 genome assembly using BWA version 0.5.9-r16 with default parameters. Reads with MAPQ no less than 30 were used for downstream analysis.
Average fragment size was esitmated from paired mapped tags. Reads 1 of paired-end reads were pooled with single-end reads of the same BioSample for downstream analysis.
Tags pileups (bedgraph files) were generated using MASC 2.0 with the following setting: -g mm --to-large --broad -f BED --nomodel --extsize 175 --buffer-size 30000000 -B --SPMR
Genome_build: mm9
Supplementary_files_format_and_content: wig files were generated using data2wig.pl from Bio-ToolBox-1.32; Scores represent tag counts per 1 million mappable reads.
|
| |
|
| Submission date |
Nov 05, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Siu Yuen Chan |
| E-mail(s) |
sychan@hku.hk
|
| Organization name |
University of Hong Kong
|
| Department |
Paediatrics & Adolescent Medicine
|
| Street address |
21 Sassoon Road
|
| City |
Hong Kong |
| ZIP/Postal code |
HK |
| Country |
Hong Kong |
| |
|
| Platform ID |
GPL11002 |
| Series (1) |
| GSE74733 |
H3K27me3 marked genes in primary hippocampal neurons |
|
| Relations |
| BioSample |
SAMN04209921 |
| SRA |
SRX1369735 |
