GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM3146354

Query DataSets for GSM3146354

Status

Public on Feb 19, 2019

Title

CEBPa_RNA-seq

Sample type

SRA

Source name

Myeloblast

Organism

Homo sapiens

Characteristics

cell type: leukemic cell line with an 8;21 chromosome translocation
cell line: Kasumi-1
passages: 4-5 passages
genotype/variation: overexpress CEBPa

Growth protocol

Kasumi-1 cells were cultured in RPMI 1640 Medium (Gibco) supplemented with 10% fetal bovine serum (FBS), 50 U/ml penicillin and 50 μg/ml streptomycin.

Extracted molecule

total RNA

Extraction protocol

At 4 days after retrovirus infection of MIGR1 and CEBPa, GFP+ Kasumi-1 cells were harvested by flow sorting and RNA was extracted with TRIzol Reagent (Invitrogen). (RNA-seq)
At 3 days after retrovirus infection of CEBPa, GFP+ Kasumi-1 cells were sorted by BD FACSARIA III through FITC channel. Then they were crosslinked with 1% formaldehyde for 10 mins and then stopped by 125 mM Glycine. After lysis and sonication, 3 μg of HA-Tag (C29F4) Rabbit mAb (CST #3724) was incubated with chromatin sample overnight at 4 ℃, and 20μl protein A/G beads (Smart lifesciences SA032005) were used for immunoprecipitation. Finally, DNA were purified with PCR recovery kit (QIAGEN #28006).(ChIP-seq)
Libraries were generated using the TruSeq RNA Sample Preparation Kit (Illumina #RS-122-2001) and sequenced with Illumina HiSeq2500.(RNA-seq)
Library preparation was according to the manufacture’s guidelines (KAPA Biosystems KK8503) and sequencing procedures were carried out according to Illumina protocols with minor modifications (Illumina, San Diego).(ChIP-seq)

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Raw reads were filtered by Seqtk(remove adapters, length less than 25 reads, ribosome RNA, Qual < 20 reads), clean sequenced reads were alignmented to hg38 by TopHat(2.0.9), allow2 mismatches and multihits <=2 per read.(RNA-seq)
Aligned reads were extracted by Htseq(0.9.1) with default parameters and normalized with TMM(trimmed mean of M values) method, FPKM were calculated by perl script.(RNA-seq)
Raw reads were filtered by Seqtk(remove adapters, length less than 25 reads, Qual < 20 reads), clean reads were alignmented to hg19 by bowtie2(2.2.9) with parameters: -N 1 -p5.(ChIP-seq)
Peaks were called by macs2(2.1.1.20160309) callpeaks algorithm with default set. BigWig files were generated by macs2 bdgcmp algorithm with default set.(ChIP-seq)
Genome_build: hg19 for ChIP-seq, hg38 for RNA-seq
Supplementary_files_format_and_content: FPKM files were calculated by definition.(RNA-seq)
Supplementary_files_format_and_content: BigWig files were generated by macs2 bdgcmp algorithm with default set.(ChIP-seq)

Submission date

May 18, 2018

Last update date

Feb 19, 2019

Contact name

Chun-hui Xu

E-mail(s)

xuch@sibs.ac.cn

Organization name

Shanghai Institute of Hematology, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine

Department

State Key Laboratory of Medical Genomics