GSM4721896: Fig 4a-5_sRNA-seq_bdf2D; Schizosaccharomyces pombe; ncRNA-Seq
Chromatin Immunoprecipitation (ChIP) experiments were performed as previously described29. Briefly, freshly grown 40 ml cells in YES were fixed with 1% formaldehyde from 37% stock (Sigma F8775) for 20 min at room temperature for H3K9me2, H3K9me3, RNA Pol II, Ago1 and Chp1 ChIP. For Swi6 and 3xFlag-Clr4 ChIP, 40 ml cells were pre-incubated at 18 °C for 2 h and fixed with 1.45% Formaldehyde from 16% stock (ThermoFisher 28908) for 30 min at 18 °C. Cells were quenched with 360 mM glycine and 2.4 mM Tris for 5 min. Whole-cell extracts (WCE) were prepared using FastPrep-24 and sonicated using Covaris. 1~2 μg of antibodies like anti-H3K9me2 (Abcam ab1220), anti-H3K9me3 (Absolute Antibody Ab00700-1.26), anti-RNA polymerase II (BioLegend 904001), Swi6 (Abcam ab188276), Ago1 (Abcam Ab18190), Chp1 (Abcam ab18191) and anti-Flag (Sigma F1804) were pre-incubated with 50 μl Protein A and Protein G sepharose (GE 17513801 and 17061801) mix for 3~4 h and then incubated with sheared chromatin overnight at 4 °C. After washing, bead-antibody-chromatin mixture were treated with Proteinase K (ThermoFisher 100005393) for 1 h ant 42 °C and incubated 5~6 h at 65 °C to reverse cross-links. DNA was cleaned up using ChIP DNA Clean & Concentrator (Zymo Research D5205) and subsequently used for qPCR or ChIP-Seq. For sRNA-seq, Total RNAs were purified from freshly grown yeast cells using Quick-RNA Fungal/Bacterial Miniprep Kit (Zymoresearch R2014) and sRNA were enriched using RNA Clean & Concentrator (Zymoresearch R1013). ChIP-Seq libraries were constructed using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB E7645) and barcoded multiplex libraries were sequenced using Illumina MiSeq platform, which generates paired-end 101 bp reads. sRNA libraries were constructed using NEBNext Small RNA Library Prep Set for Illumina (NEB E7330) and barcoded multiplex libraries were sequenced using Illumina MiSeq platform, which generates single-end 36 nt reads.
GEO Accession
GSM4721896