|
| Status |
Public on Aug 02, 2020 |
| Title |
KO_input |
| Sample type |
SRA |
| |
|
| Source name |
Setd2 knockout KPC1199 cells using CRISPR/Cas9, as KO
|
| Organism |
Mus musculus |
| Characteristics |
genotype/variation: Setd2 knockout using CRISPR/Cas9
cell line: KPC1199
antibody: IgG
|
| Treatment protocol |
KPC1199 cells were infected with lentivirus expressing both Cas9 enzyme and sgRNA against LacZ and Setd2, respectively. Monoclonal cells were selected with 2μg/ml puromycin treatment for 2 weeks.
|
| Growth protocol |
KPC1199 cells were cultured in DMEM containing 10% FBS and 1% penicillin/streptomycin
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
RNA was harvested using Trizol reagent (Invitrogen). Chromatin was prepared from isolated acinar cells or cell lines using standard protocol of ChIP Kit (Active Motif). In brief, cells were fixed by 1% formaldehyde for 10 min and terminated by glycine for 5 min (final concentration = 0.125 M). After 2 times washing by pre-cooling PBS (protease inhibitor containing), cells were scraped and resuspended in SDS-lysis buffer respectively (50 mM Tris-HCl Ph 7.5, 10 mM EDTA, 1% SDS and protease inhibitor), and sonicated to shear crosslinked DNA to about 200-bp. Fifty micrograms of fragmented chromatin was incubated overnight with 2.5 μl of anti-H3K36me3 antibody (Abcam, ab9050) and normal IgG (used as negative control). Complexes were isolated using pre-cleaned protein G agarose (1 hour at 4 ℃). Immunoprecipitated DNA was prepared as a standard illumine library according to Illumina's instructions.
libraries were prepared for sequencing using standard Illumina protocols
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
HiSeq X Ten |
| |
|
| Description |
KO_IPvsKO_input_peaks.bed
|
| Data processing |
Illumina Casava1.7 software used for basecalling.
We applied the reads filtering towards the raw reads after sequencing to achieve the clean data following the criteria: a)10% base quality <15 b) 13% base quality <20.
RNA:Clean Reads mapped to reference genome using Hisat2 v2.0.4
RNA:count counting
ChIP: Clean Reads mapped to reference genome using bowtie2
ChIP: peaks were called using macs2
Genome_build: GRCm38
Supplementary_files_format_and_content: RNA:TXT file contain counts for each sample
Supplementary_files_format_and_content: ChIP:bed files were generated using macs2
|
| |
|
| Submission date |
Feb 08, 2019 |
| Last update date |
Aug 02, 2020 |
| Contact name |
Ningning Niu |
| E-mail(s) |
niunn2014@outlook.com
|
| Phone |
86-21-68382281
|
| Organization name |
Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital
|
| Department |
Clinical Stem Cell Research Center
|
| Street address |
160 Pu Jian Road, Shanghai
|
| City |
Shanghai |
| ZIP/Postal code |
200127 |
| Country |
China |
| |
|
| Platform ID |
GPL21273 |
| Series (1) |
| GSE126302 |
Loss of Setd2 promote Kras-induced acinar-to-ductal metaplasia and epitheliamesenchymal transition during pancreatic carcinogenesis |
|
| Relations |
| BioSample |
SAMN10890114 |
| SRA |
SRX5352362 |
