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| Status |
Public on Mar 09, 2017 |
| Title |
ChrRNA-seq-DGCR8KO - replicate 2 |
| Sample type |
SRA |
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| Source name |
DGCR8 knockout embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: Embryonic stem cells
genotype: DGCR8-KO
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| Treatment protocol |
Cells were treated with DMSO or JQ1 (500 nM) for 12 hrs before fractionation.
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| Growth protocol |
mESCs were cultured in mES media.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were trypsinized, washed with cold PBS and resuspended in cold lysis buffer (10 mM Tris pH 7.4; 150 mM NaCl; 0.15% NP-40; RNase inhibitor). After 5 min incubation on ice, the suspension was layered over 1 ml sucrose buffer (10 mM Tris pH 7.4; 150 mM NaCl; 24% sucrose) and centrifuged (5 min, 3500 g). After removal of supernatant, nuclei pellet was briefly rinsed with cold PBS, resuspended in 250 μl Glycerol buffer (20 mM Tris pH 7.4; 75 mM NaCl; 0.5 mM EDTA; 50% Glycerol) and mixed with 250 μl nuclear lysis buffer (10 mM Tris pH 7.4, 7.5 mM MgCl2, 0.2 mM EDTA, 0.3 M NaCl, 1 M Urea, 1% NP-40) by vortexing for 4 sec. After 2 min incubation on ice, samples were centrifuged for 2 min at full speed. The remaining chromatin pellet was rinsed with cold PBS and resuspended in QIAzol lysis agent. RNA was extracted using QIAshredder and miRNeasy Mini Kit (Qiagen).
After removal of rRNA by Ribo-Zero rRNA removal kit (Illumina), DNA libraries were generated using TruSeq Stranded Total RNA LT Sample Prep Kit and analyzed by NextSeq.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
chromatin-associated RNA
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| Data processing |
Library strategy: ChrRNA-seq
ChrRNA-seq samples were analyzed by NextSeq.
Reads were mapped to mm9 assembly and ChrRNA abundance at gene level was estimated using TopHat (version 2.0.12.) and Cufflinks (version 2.2.0.) (Trapnell et al., 2012).
For quantification of pri-miRNAs, reads were mapped to mm9 assembly with bowtie 1.0.1 (Langmead et al., 2009), allowing two mismatches, and quantitated as the numbers of reads that mapped to pre-miRNA and flanking 100 base pair regions in a reads per million scale.
Genome_build: mm9
Supplementary_files_format_and_content: Bw files were produced in a rpm scale.
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| Submission date |
Nov 14, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Hiroshi I. Suzuki |
| E-mail(s) |
hisuzuki-tky@umin.ac.jp
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| Organization name |
Nagoya University
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| Department |
DIvision of Molecular Oncology, Graduate School of Medicine
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| Street address |
65 Tsurumai-cho, Showa-ku
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| City |
Nagoya |
| ZIP/Postal code |
466-8550 |
| Country |
Japan |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE89821 |
Super-enhancer-mediated RNA processing revealed by integrative microRNA network analysis [ChrRNA-seq] |
| GSE89826 |
Super-enhancer-mediated RNA processing revealed by integrative microRNA network analysis |
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| Relations |
| BioSample |
SAMN06014901 |
| SRA |
SRX2345846 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2390060_ChrRNA_DGCR8KO_rep2.bw |
435.8 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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