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Sample GSM2062751

Query DataSets for GSM2062751

Status

Public on Sep 01, 2016

Title

leukemia_T2F3_2_mpp_RNASeq

Sample type

SRA

Source name

bone marrow

Organism

Mus musculus

Characteristics

strain: C57BL/6
technology: RNA-Seq
cell type: MPP
genotype: Tet2-/-Flt3ITD

Growth protocol

mESCs were cultured in feeder-free gelatin-coated plates in Dulbecco’s Modified Eagle Medium (DMEM) (Invitrogen Cat. No. 11995) supplemented with 15% FBS (GIBCO), 2 mM L-glutamine (GIBCO), 0.1 mM 2-mercaptoethanol (Sigma), 1 × nonessential amino acids (GIBCO), 1,000 units/ml LIF (Millipore Cat. No. ESG1107), 1 × pen/strep (GIBCO), 3 mM CHIR99021 (Stemgent), and 1 mM PD0325901 (Stemgent). Half of the medium was changed every day for 5 days before harvesting mEBs by sedimentation. Hematopoietic stem and progenitor cells were harvested from sacrificed animals treated as described in the methods section of the associated manuscript. Cell surface phenotypes were LSK (lin- Sca+ cKit+xxx markers), MPP (lin- Sca+ cKit+ CD48+ CD150-), CMP (lin- Sca- cKit+ CD34+ CD16/32-), MEP (lin- Sca- cKit+ CD34- CD16/32-), GMP (lin- Sca- cKit+ CD34+ CD16/32+).

Extracted molecule

total RNA

Extraction protocol

DNA and RNA were isolated using the AllPrep DNA/RNA Mini Kit from Qiagen.
The tagmentation reactions were performed in a 50μl solution with 1X tagmentation buffer from Nextera DNA sample preparation kit (FC-121-1031). The input DNA is ranged from 0.5 ng to 50 ng. The reactions were performed according to the manufacturer’s instruction. The glucosylation reactions were performed in a 20 μl solution containing 50 mM HEPES buffer (pH 8.0), 25 mM MgCl2, purified DNA, 100 μM N3-UDP-Glc, and 1 μM βGT, at 37°C for 1 hr. After that, DBCO-PEG4-DBCO (Click Chemistry Tools, 20 mM stock in DMSO) was added to the reaction mixture. The reactions were incubated at 37°C for 2 hr. Next, the DNA was purified by Micro Bio-Spin 30 Column (Bio–Rad). The purified DNA incubated with 5 µL C1 Streptavidin beads (Life Technologies) in 2X buffer (5 mM Tris pH 7.5, 0.5 mM EDTA, 1 M NaCl) for 15 min. The beads were subsequently undergone six 5-min washes each with 1X buffer. All binding and washing were done at room temperature with gentle rotation. The captured DNA fragments were amplified with 12-18 cycles of PCR amplification using the enzyme mix supplied by the Nextera kit. The PCR products were purified using AMPure XP beads. Separate input libraries were made by direct PCR from fragmented DNA without labeling and capture.RNA-seq libraries are constructed by True-seq stranded mRNA sample preparation kit (Illumina). The reaction is performed according to the manufacture’s instruction. Sequencing was performed on the Hi-Seq instrument.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Data processing

For nano-hmC-Seal, Illumina reads were post-processed and aligned to the mouse mm9 assembly using the bowtie program with default parameters.
To visualize sequencing signals in the genome browser, we generated bedGraph files with HOMER
For RNA-Seq,sequencing reads were aligned to mm9 genome by STAR. Count matrices were generated by summarizeOverlaps functionality from R package GenomicAlignments. The rlog transformation were then performed by the R package "DESeq2"
Genome_build: mm9
Supplementary_files_format_and_content: For nano-hmC-Seal sample, bedGraph files for each dataset was generated by HOMER software.
Supplementary_files_format_and_content: For RNA-Seq samples, "RNASeq_rld_expr.txt" contain rlog transfomed counts per Gene.

Submission date

Feb 16, 2016

Last update date

May 15, 2019

Contact name

Dali Han

E-mail(s)

[email protected]

Organization name

University of Chicago

Department

Department of Chemisty