GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM2461449

Query DataSets for GSM2461449

Status

Public on Apr 19, 2017

Title

ALYREF-RIP-rep1

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 rep1.

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

E-mail(s)

sunbf@big.ac.cn

Organization name

Beijing Institute of Genomics (BIG) of Chinese Academy of Sciences (CAS)

Street address

Da-Tun Road