 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Sep 01, 2016 |
| Title |
hematopoiesis_cmp_1 |
| Sample type |
SRA |
| |
|
| Source name |
bone marrow
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
technology: nano-hmC-Seal
cell type: CMP
genotype: wild-type
|
| 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 |
genomic DNA |
| 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 |
MeDIP-Seq |
| Library source |
genomic |
| Library selection |
5-methylcytidine antibody |
| 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
|
| Street address |
5801 South Ellis Avenue
|
| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60637 |
| Country |
USA |
| |
|
| Platform ID |
GPL13112 |
| Series (1) |
| GSE77967 |
A highly sensitive and robust method for genome-wide 5hmC profiling of rare cell populations |
|
| Relations |
| SRA |
SRX1586801 |
| BioSample |
SAMN04501994 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2062721_hematopoiesis_cmp_1.ucsc.bedGraph.gz |
16.6 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
