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Sample GSM3574395

Query DataSets for GSM3574395

Status

Public on Jan 23, 2019

Title

Input_rep1

Sample type

SRA

Source name

embryonic stem cells

Organism

Mus musculus

Characteristics

strain: C57BL/6(F) x 129/sv(M)
developmental stage: 3.5 days
cell type: embryonic stem cells
antibody: none

Treatment protocol

For PD+LIF experiments, ESCs were first plated in 2i+LIF for 24 hr, then washed once with 1xPBS and changed to N2B27 supplemented with PD+LIF, cultured for another 4 days before analysis with medium changed every other day.For RIP-seq, 3XFLag-Trim71 overexpressing and empty ESCs cultured in 2i+LIF medium were UV-crosslinked and lysed in lysis buffer. 1/40 lysis was taken as input. The remaining lysis was subjected to immunoprecipitation using FLAG antibody.

Growth protocol

Mouse ESCs were cultured on 0.1% gelatin-coated plates in 2i+LIF medium which is consist of N2B27 supplemented with MEK inhibitor PD0325901 (1.0 μM), GSK3 inhibitor CHIR99021 (3.0 μM) and leukemia inhibitory factor (1,000 unit /ml).

Extracted molecule

total RNA

Extraction protocol

For RNA-seq,cells were treated with TRIzol and followed with phase seperation, RNA precipitation and RNA wash. Finally, RNA were dissolved in DEPC water.For RIP-seq,The RNA in input lysis and precipitated beads were extracted by TRIzol.
For RNA-seq,total RNA was subjected to two rounds of purification using poly-T oligo-attached magnetic beads before the synthesis of double-stranded (ds) cDNA. The ds cDNA was ligated to adaptors and sequenced with illumina Hiseq2000 platform.For RIP-seq,Input RNA were treated as the total RNA in RNA-seq,the IP RNA RNA from RIP experiment was directly used for the synthesis of ds-cDNA.

Library strategy

RIP-Seq

Library source

transcriptomic

Library selection

other

Instrument model

Illumina HiSeq 2000

Description

replicate1
Trim71_RIP.txt

Data processing

Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to mm9 whole genome using STAR v2.5.0
Fragments Per Kilobase of exon per Megabase of library size (FPKM) were calculated using Cufflinks v2.2.1
For RIP-seq,reads aligned to non-polyA transcripts were excluded. To calculate fold enrichment for RIP, we normalized the FPKM value of every transcript to the average of all transcripts.
Genome_build: mm9
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample in txt file

Submission date

Jan 22, 2019

Last update date

Jan 26, 2019

Contact name

Yangming Wang

E-mail(s)

yangming.wang@pku.edu.cn

Phone

0118610-62766945

Organization name

Peking University