|
| Status |
Public on Nov 26, 2018 |
| Title |
WT+rnaseH BOBSC (negative control) |
| Sample type |
SRA |
| |
|
| Source name |
human iPS cell line, spiked in with DT40 cell line
|
| Organisms |
Gallus gallus; Homo sapiens |
| Characteristics |
cell line: BOBSC iPS cell line
genotype/variation: wild type
antibody: S9.6
spiked with: chicken DT40 cell line
molecule subtype: nuclear RNA
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Upon mixing the 50-60 million BOBSC iPS cells with the DT40 cells (10:1 ratio) , the nuclear fraction was extracted (then treated with rnaseH for the control), genomic DNA containing containing DNA:RNA hybrid purified, soluble contaminating RNA was digested with RNase I, sonicated to average size of 300 bp with Bioruptor Diagenodtreatments and immunoprecipitated DNA:RNA hybrids with S9.6 antibody. Following low, high, LiCl and TE buffer washes of Protein-G captured immunocomplexes, DNA:RNA hybrids were eluted, phenol:chloroform purified, denatured for 5 minutes at 95 C, DNA moity digested with DNaseI, and RNA moiety of the DNA:RNA hybrid purified with Trizol.
Directional RNA libraries were prepared using NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs, E7645) per instructions provided. Steps from random priming to End Prep of cDNA was followed as instructed in for rRNA depleted FFPR RNA samples, and further steps where followed as instructed for purified mRNA or rRNA depleted RNA.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
SLX-13376.NEBNext09.HNMG2BBXX.s_1.r_1
|
| Data processing |
Reads were quality trimmed and filtered for adaptor sequences using trim galore v0.4.4
Reads were aligned to Ggal 5.0 and GRCh38 using bowtie2 v2.2.6
Uniquely mapping reads were separated into those aligning to the sample and the spike-in
Peaks were called using MACS2 v2.1.1.20160309 with options -g 1.87e9 –broad
Genome_build: Ggal 5.0 and GRCh38
Supplementary_files_format_and_content: Tab-delimited files produced by MACS2 containing processing metadata and per peak information
|
| |
|
| Submission date |
Apr 05, 2018 |
| Last update date |
Nov 26, 2018 |
| Contact name |
Alastair Crisp |
| Organization name |
MRC Laboratory of Molecular Biology
|
| Street address |
Francis Crick Avenue
|
| City |
Cambridge |
| ZIP/Postal code |
CB2 0QH |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL24850 |
| Series (2) |
| GSE112745 |
Genome wide mapping of R-loops in the repriming deficient human cells using RNA DIP-Seq |
| GSE112747 |
Repriming limits R-loop formation at DNA secondary structures during S phase |
|
| Relations |
| BioSample |
SAMN08871780 |
| SRA |
SRX3892923 |
