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Sample GSM3505643

Query DataSets for GSM3505643

Status

Public on Feb 05, 2019

Title

Foxp3Sf_Th2-RA_r1

Sample type

SRA

Source name

Rag2 OT-II Foxp3Sf (Scurfy) Th2 cells

Organism

Mus musculus

Characteristics

strain: C57BL/6
tissue: Spleen and lymph nodes
culture condition: in vitro culture for 72 hours with anti CD3/28 and Th2-promoting cytokines/antibodies
chip antibody: N/A

Extracted molecule

total RNA

Extraction protocol

For in vitro polarized cells, total RNA was prepared from approximately 500,000 cells using an Ambion mirVana miRNA isolation kit following the manufacturer’s protocol (ThermoFisher AM1560). For in vivo IL-9 producing cells and other subsets, total RNA was prepared from approximately 2,000-10,000 cells using a Trizol-based extraction technique following the manufacturer’s protocol (ThermoFisher 15596026).
Total RNA was subsequently processed to mRNA-seq library using NEBNext Poly(A) mRNA Magnetic Isolation followed by either Ultra II Directional RNA Library Prep kit for Illumina (NEB E7490, E7760, E7335); or Mondrian Ovation Ultralow according to the vendor’s manual for the Illumina platform (Cat#0344; NuGEN).
The purified libraries were multiplexed and captured on an Illumina flow cell for cluster generation. Libraries were sequenced for 50 single read cycles on HiSeq 2500 following the manufacturer's protocols.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

150428_0000_b_s11
RNA-Seq_R23-R70_rpkm.xlsx
R37

Data processing

base calling was carried out using CASAVA1.8.2/bcl2fastq1.8.4
ATAC-seq, alignment: PE reads were mapped to the mouse genome (mm9 assembly) using Bowtie 1.1.1.
ATAC-seq, peak calling: Redundant reads were removed using Fastquniq. Uniquely mapped reads from fragments less than 175 bp were used to call peaks with MACS1.4.2 using a p-value threshold of 1 x 10-5
RNA-seq, alignment: SE reads were mapped to the mouse genome mm9 using TopHat 2.1.0
RNA-seq, RPKM calculation RNA-seq: RPKMs were calculated using Partek6.6
ChIP-seq, alignment: SE reads were aligned to the mouse genome build mm9 with Bowtie 1.1.1
ChIP-seq, peak calling: Non-redundant and Uniquely mapped reads were used to call peaks with MACS1.4.2 using a p-value threshold of 1 x 10-5
Genome_build: mm9
Supplementary_files_format_and_content: peak bed files for ATAC-seq & ChIP-seq generated by MACS1.4.2; RPKM files for RNA-seq generated by Partek 6.6

Submission date

Dec 07, 2018

Last update date

Feb 05, 2019

Contact name

Hong-wei Sun

Organization name

NIAMS

Department

Office of Science & Technology

Lab

Biodata Mining & Discovery Section