|
| Status |
Public on Apr 12, 2017 |
| Title |
siRNA_mlo3Δ |
| Sample type |
SRA |
| |
|
| Source name |
mlo3Δ
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
antibody for ip: anti-FLAG
strain number: 558
target molecule: siRNA
|
| 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.
Endogenous 3xFLAG-tagged Ago1-bound siRNA were purified by protein affinity purification and subsequent size selection on an 18% acrylamide gel. 2 pmol of a preadenylated 3' adaptor oligonucleotide (miRNA Cloning Linker-1 from IDT, 5'-App CTG TAG GCA CCA TCA AT/ddC/-3') were ligated in a 10 µl reaction with 5 U T4 RNA ligase (TaKaRa), ligation buffer without ATP and 5 U RNasin (Promega) at 20°C for 2 hours. The 3' ligated products were purified on an 18% acrylamide urea gel with subsequent phenol-chloroform purification and ethanol purification. The 5' adaptor ligation was performed in a 10 µl reaction with 2 pmol 5' adaptor oligonucleotide (5'-GUU CAG AGU UCU ACA GUC CGA CGA UC-3'), 5 U RNasin (Promega), 0.06 µg BSA, 5 U T4 RNA ligase (Thermo Scientific) and 1x ligation buffer with ATP (Thermo Scientific) for 2 h at 20°C. The ligated products were gel purified and reverse transcribed with 10 pmol primer (RT primer: 5'- GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC GAT TGA TGG TGC CTA CAG-3') and the SuperScript III First Strand Synthesis System (Thermo Scientific). The cDNA was PCR-amplified with Q5 High-Fidelity 2x Master Mix (NEB) for 14-20 cycles using the Illumina P5 5' primer (5' -AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC G -3') and the Illumina P7 3' primer with inserted barcode (5'-CAA GCA GAA GAC GGC ATA CGA GAT XXXXXX GTG ACT GGA GTT CAG ACG TG -3').
|
| |
|
| Library strategy |
ncRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| 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 |
GPL9453 |
| Series (1) |
| GSE94129 |
Accumulation of RNA on chromatin disrupts heterochromatic silencing |
|
| Relations |
| BioSample |
SAMN06275897 |
| SRA |
SRX2522338 |
