GSM5419609: S. pombe, clr4del_nascentRNA_10min_3; Schizosaccharomyces pombe; RNA-Seq
ChIP libraries: IP of crosslinked (1% formaldehyde, 15 min) sonycated lysate, followed by decrosslinking and protein and RNA degradation. DNA library construction with NEBNext Ultra II DNA Library Prep Kit for Illumina kit (NEB). Nascent RNA: RNA was labeled with 4 Thiouracil (Sigma), extracted, after removal of DNA was treated EZ-Link HPDP-Biotin (Thermo Scientific). Labeled RNA was recovered using streptavidin beads (Roche). RNA libraries were prepared with NEBnext Ultra Directional RNA Library Prep Kit for Illumina (NEB). Total RNA was isolated applying the hot phenol method. The extracted nucleic acids were treated with DNAse I, which was removed by a second phenol-chloroform-isoamylalcohol extraction and ethanol precipitation. RNA libraries were prepared with NEBnext Ultra Directional RNA Library Prep Kit for Illumina (NEB). Small RNA libraries: 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'). DNA and RNA libraries were prepared using NEBnext Ultra Directional RNA or DNA Ultra II Library Prep Kit for Illumina (NEB), following the manufacturer instruction. 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.