|
| Status |
Public on Apr 12, 2017 |
| Title |
Caf1RIP_chr_untagged 1 |
| Sample type |
SRA |
| |
|
| Source name |
wild type
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
antibody for ip: anti-FLAG M2-Magnetic Beads (Sigma-Aldrich)
strain number: 63
target molecule: background RNA
|
| Growth protocol |
cells were grown in liquid, rich YES medium
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA libraries were prepared with NEBnext Ultra Directional RNA Library Prep Kit for Illumina (NEB); DNA library construction with NEBNext Ultra II DNA Library Prep Kit for Illumina kit (NEB). siRNA library: ligation of 3’ adapter (5'-App CTG TAG GCA CCA TCA AT/ddC/-3') and 5’ adapter (5'-GUU CAG AGU UCU ACA GUC CGA CGA UC-3'), reverse transcription, PCR of cDNA with Illumina P5 5' primer and barcoded llumina P7 3' primer.
Crosslinked cells with 1% Formaldehyde for 15 minutes used. Caf1-associated RNA IP with chromatin fractionation: before sonication, the sample was centrifuged for 20 min at 21 000 x g at 4°C. The supernatant was taken as “soluble fraction”, the pellet was washed twice with lysis buffer, then resuspended in lysis buffer, which formed the “chromatin fraction”. Subsequent immunoprecipitation, decrosslinking, protein degradation and DNA depletion.
|
| |
|
| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 1500 |
| |
|
| Data processing |
base calling with RTA1.18.64
Sequenced reads were trimmed for the adaptor sequence, then mapped to the S.pombe genome with Novoalign (http://www.novocraft.com) randomly assigned.
Reads mapping with two or fewer mismatches were retained.
Enrichments were calculated with our house Perl scripts
Normalisation to reads per million for siRNA and ChIP experiments, normalisation to reads mapping to protein coding genes for RNAseq /RIPseq
Genome_build: Schizosaccharomyces_pombe.ASM294v1.18
Supplementary_files_format_and_content: can be viewed with IGV browser: http://www.broad.mit.edu/igv
|
| |
|
| Submission date |
Jan 27, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Mario Halic |
| E-mail(s) |
halic@genzentrum.lmu.de
|
| Organization name |
Ludwig-Maximilians-Universität
|
| Department |
Biochemistry - Gene Center
|
| Lab |
Halic
|
| Street address |
Feodor-Lynen-Str.25
|
| City |
Munich |
| ZIP/Postal code |
81377 |
| Country |
Germany |
| |
|
| Platform ID |
GPL22681 |
| Series (1) |
| GSE94129 |
Accumulation of RNA on chromatin disrupts heterochromatic silencing |
|
| Relations |
| BioSample |
SAMN06275871 |
| SRA |
SRX2522296 |
