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Sample GSM2470620 Query DataSets for GSM2470620
Status Public on Apr 12, 2017
Title siRNA_taz1Δ
Sample type SRA
 
Source name taz1Δ
Organism Schizosaccharomyces pombe
Characteristics antibody for ip: anti-FLAG
strain number: 633
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 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 SAMN06275901
SRA SRX2522335

Supplementary file Size Download File type/resource
GSM2470620_siRNA_taz1D_minus.igv.tdf 710.4 Kb (ftp)(http) TDF
GSM2470620_siRNA_taz1D_plus.igv.tdf 707.3 Kb (ftp)(http) TDF
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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