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Sample GSM2461450 Query DataSets for GSM2461450
Status Public on Apr 19, 2017
Title ALYREF-RIP-rep2
Sample type SRA
Source name Human HeLa cells
Organism Homo sapiens
Characteristics cell line: HeLa
rip antibody: ALYREF (Sigma, Cat# A2220, lot# SLBN7698V)
age: P7
tissue: carcinoma cervical
Extracted molecule total RNA
Extraction protocol Flag-ALYREF overexpressed cells pellets were resuspended with 2 volume of lysis buffer (150 mM KCl, 10 mM HEPES pH 7.6, 2 mM EDTA, 0.5% NP-40, 0.5 mM DTT, 1:100 protease inhibitor cocktail, 400 U/ml RNase inhibitor), and incubated at 4 °C for 30 min with rotation. Then the lysate was centrifuged at 15,000 g for 20 min. The anti-Flag M2 magnetic beads (Sigma, 10 µl per mg lysate) was washed with a 600 ml NT2 buffer (200 mM NaCl, 50 mM HEPES pH 7.6, 2 mM EDTA, 0.05% NP-40, 0.5 mM DTT, 200 U/ml RNase inhibitor) four times and then resuspended in 800 ml ice-cold NT2 buffer. Cell lysate was mixed with M2 beads and incubated at 4 °C for 4 hr with rotation. The beads were washed two times with 1 ml ice-cold NT2 buffer. Then the beads were subject to Micrococal nuclease (NEB) digestion (1:1,000,000 dilution) for 8 min at 37°C. The beads were cooled on ice immediately for 5 min and washed two times with 1 ml ice-cold 1× PNK+EGTA buffer (50 mM Tris-HCl pH 7.5, 20 mM EDTA, 0.05% NP-40, 200 U/ml RNase inhibitor) and two times with 1 ml ice-cold 1× PK buffer (50 mM NaCl, 100 mM Tris-HCl pH 7.5, 10 mM EDTA, 0.2% SDS, 200 U/ml RNase inhibitor). Then the beads were digested with 200 μl pre-heated (20 min at 50°C) Proteinase K solution (4 mg/ml) for 40 min at 50°C with rotation at 2000 rpm/min. After centrifuge at top speed for 5 min, the supernatant was transferred to a new 1.5 ml tube and isolated the RNAs with equal volume Acid-Phenol: Chloroform, pH 4.5 (Ambion). The RNAs were subjected to RIP seq.
Approximately 2.5 µg of total RNA was then used for library preparation using a TruSeq™ RNA Sample Prep Kit v2 (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol.The libraries were sequenced using HiSeq2500 in paired-read mode, creating reads with a length of 125 bp.
Library strategy RIP-Seq
Library source transcriptomic
Library selection other
Instrument model Illumina HiSeq 2500
Description ALYREF RIP-seq of normal HeLa cells rep2.
Data processing Reads were aligned to hg19 genome using TopHat v2.0.9.
The ALYREF binding regions (peaks) were identified by using the MACS2 (version 2.0.10) with p value < 10-5.
Genome_build: hg19.
Submission date Jan 18, 2017
Last update date May 15, 2019
Contact name Bao-Fa Sun
Organization name Beijing Institute of Genomics (BIG) of Chinese Academy of Sciences (CAS)
Street address Da-Tun Road
City Beijing
ZIP/Postal code 100101
Country China
Platform ID GPL16791
Series (2)
GSE93750 Transcriptomics of siCTRL, siNSUN2 and ALYREF-RIP HeLa cells, and multiple mouse tissues
GSE93751 5-methylcytosine promotes mRNA export
BioSample SAMN06237539
SRA SRX2499552

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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