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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Dec 31, 2020 |
| Title |
scMNase-seq_EILP_GC0416 |
| Sample type |
SRA |
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| Source name |
EILP
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
genotype/variation: Tcf7EGFPnull/+
tissue: bone marrow
sorting profile: Lin- Kit+ 2B4+ a4b7+ Tcf7+ Thy1-
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| Growth protocol |
Samples were isolated by cell sorting from bone marrow single cell suspensions
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For scMNase-Seq, single live cells for EILP, EILPTcf7-/-, ILCP were sorted by flow cytometer and deposited directly into 96 well plate, which containing 32 μl of cell lysis buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 2 mM CaCl2, 0.1% Triton X-100). 32 μl of cell lysis buffer. 8 μl diluted MNase (1:3000 in lysis buffer) was added to the sorted cells and incubated at 37 °C for 5 minutes. The reaction was stopped by addition of 80 μl of stop buffer (10mM Tris-HCl, pH 7.5, 10mM NaCl, 0.15% SDS, 10mM EGTA), and 1 μl of 20 mg/ml Proteinase K. Following incubation at 65°C for 2 hours, DNA was purified by Phenol-chloroform extraction and precipitation with ethanol in the presence of glycogen.
For MNase-seq libraries, DNA were end-repaired in 50 μl of reaction volume at RT for 45 minutes using End-It DNA-Repair kit (Epicenter, Cat#ER81050) according to the manufacturer’s instructions. Next, DNA were purified by enzyme reaction clean kit (Qiagen). The blunted DNA fragments were subject to adding 3 end A base using the Klenow fragment (3′ → 5′ exo-) (NEB). The DNA was then ligated with the ‘‘Y’’-shaped Illumina adaptor and amplified for 16 cycles using indexing primers with the PCR condition: 98°C,10”; 67°C, 30”; 72°C, 30”. PCR products between 150-250 bp were isolated on 2% E-gel for sequencing on Illumina Hiseq-3000.
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| Library strategy |
MNase-Seq |
| Library source |
genomic |
| Library selection |
MNase |
| Instrument model |
Illumina HiSeq 3000 |
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| Data processing |
Basecalling by RTA 1.18.66.3, Illumina instrument run time analysis software
Sequencing reads were aligned to mouse genome (mm9) using Bowtie2 with default parameters.
Reads from replicates were merged and redundant reads were removed from further analysis.
For ChIC-seq samples, reads from replicates were merged for peak calling. Peak calling were performed using MACS2 with default parameters.
For scMNase-seq samples, reads with length between 140 and 180 bp were selected as nucleosome reads while reads with length less than 80 bp were selected as subnucleosomal reads.
Genome_build: mm9
Supplementary_files_format_and_content: For ChIC-seq and DNase-seq samples, .wig files contain normalized tag density, which can be uploaded to UCSC genome browser for visulization. For scMNase-seq samples, bed files provide genomic positions on chromosomes for nucleosomes (140-180 bp) and subnucleosomes (<80 bp).
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| Submission date |
Dec 20, 2019 |
| Last update date |
Jan 01, 2021 |
| Contact name |
Keji Zhao |
| Organization name |
NHLBI,NIH
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| Department |
Systems Biology Center
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| Lab |
Laboratory of Epigenome Biology
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| Street address |
9000 Rockville Pike
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platform ID |
GPL21493 |
| Series (1) |
| GSE142468 |
Transcription factor TCF1 is indispensable for the epigenetic priming of EILPs toward distinct cell fates |
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| Relations |
| BioSample |
SAMN13669933 |
| SRA |
SRX7429134 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4229277_GC0416.140_180.bed.gz |
431.9 Kb |
(ftp)(http) |
BED |
| GSM4229277_GC0416.les80.bed.gz |
101.0 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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