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Sample GSM4987885

Query DataSets for GSM4987885

Status

Public on May 03, 2021

Title

SGC7901_α-ILF3_target

Sample type

SRA

Source name

gastric cancer cell

Organism

Homo sapiens

Characteristics

cell line: SGC7901
cell type: gastric cancer
group: Target (anti-ILF3)

Treatment protocol

After crosslinking with 0.5% formaldehyde for 10 min at room temperature, cells were harvested and lysed in RIP lysis buffer with RNasin (1000 U/ml), DNase I (50 U/ml) and protease inhibitor cocktail. After the genomic DNA was digested, lysates were further subjected to sonication. Supernatants cleared by centrifugation were incubated with the anti-ILF3 antibody or IgG overnight at 4 °C. Protein A/G beads were added for a further 4 h incubation at room temperature. After the beads were washed with wash buffer, immunocomplexes of proteins and RNAs were de-crosslinked at 95 °C for 15 min. The immunoprecipitated RNAs were then purified for RNA sequencing and qRT-PCR analysis.

Growth protocol

The human gastric cancer cell line SGC7901 was purchased from the Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China). The gastric cancer cell line SGC7901 was cultured in DMEM medium containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 U/mL streptomycin and 0.03% glutamine at 37 oC in 5% CO2.

Extracted molecule

total RNA

Extraction protocol

The immunoprecipitated RNA was extracted using Trizol reagent (Invitrogen, USA) according to the manufacturer’s instructions. The RNA purity and concentration was checked using the NanoPhotometer spectrophotometer (IMPLEN, CA, USA).
RNA samples were fragmented into fragments by RNA fragmentation buffer. First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 250~300 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 µl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37℃ for 15 min followed by 5 min at 95℃ before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.

Library strategy

RIP-Seq

Library source

transcriptomic

Library selection

other

Instrument model

Illumina HiSeq 4000

Data processing

Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyses were based on the clean data with high quality.
Reference genome and gene model annotation files were downloaded from genome website directly. Paired-end clean reads were aligned to the reference genome using Bowtie v2.2.6.
After mapping reads to the reference genome, we used the MACS2 version 2.1.0 (model-based analysis of ChIP-seq) peak finding algorithm to indentify regions of IP enrichment over background. A q value threshold of enrichment of 0.05 was used for all data sets. Then, the distribute of chromosome distribution, peak width, fold enrichment, significant level and peak summit number per peak were all displayed.
Genome_build: GRCh38
Supplementary_files_format_and_content: tab-delimited peak file include information about identified peaks

Submission date

Dec 24, 2020

Last update date

May 03, 2021

Contact name

Qin Shanshan

E-mail(s)

[email protected]

Organization name

Institute of Basic Medical Sciences, School of Basic Medical Sciences, Hubei University of Medicine

Street address

Renmin Road 30, Maojian District