title: GSM7503479: P6-mDRIP-seq-def1Δ-Input-R2; Saccharomyces cerevisiae; OTHER
library_construction_protocol: For mammal and E. coli genomic DNA extraction, cells were lysed by 0.5% SDS and digested with 0.1mg/mL proteinase K (TransGen Biotech#: GE201-01) at 37°C in a shake with 220 rpm for 4-6 hours followed by phenol-chloroform nucleic acid extraction method. For Arabidopsis and rice gDNA extraction, seedlings were grounded in liquid nitrogen and then lysed by 0.5% SDS and digested with 0.1 mg/mL proteinase K (TransGen Biotech#: GE201-01) at 37°C in a shake with 220 rpm for 4-6 hours followed by phenol-chloroform nucleic acid extraction method. For yeast gDNA extraction, cells were resuspended in TE buffer with 0.5% NP40 and equal volume glass beads (Sigma#: G8772), and beat 6 times in 30 sec at 60 Hz with 1 min pausing at 4°C to destroy cell walls. Then cells were collected, lysed by 0.5% SDS and digested with 0.1mg/mL proteinase K at 37°C in a shake with 220 rpm for 4-6 hours followed by phenol-chloroform nucleic acid extraction method. For cDNA hybrids synthesis, total RNA was extracted according to the manual of TRIzol™ (ThermoFisher Scientific#:15596026) from the manufacturer. DNase I (ThermoFisher Scientific#: EN0521) treatment was performed at 37°C to remove potential gDNA contamination. cDNA hybrids were synthesized by ProtoScript® II Reverse Transcriptase with d(T)23VN primer according to the manufacturer's manual. Then, cDNA hybrids were incubated with MBN at 30°C for 30 min to remove remaining primers and RNA while only production with DNA:RNA hybrid form can be retained. Nucleic acid was digested with MBN at 30°C for 1 hour and purified using the phenol-chloroform nucleic acid extraction method followed by resuspending the pellet in 130 μl TE buffer. Sonication with Covaris™ ME220 (Covaris, 70 W, 20% Duty factor, 1000 cycles per burst, 130 s, at 4–20°C) was performed to fragment nucleic acid with peak size around 250 bp and quantified by Qubit™ dsDNA HS Assay Kit (ThermoFisher Scientific#:Q32851). For multiple samples that would be used as a pool for DRIP, sonicated DNA was diluted to the needed concentration and barcoded in DNA:RNA hybrids tailing and ligation reaction for each sample as above mentioned at 37°C for 1 hour with barcode adaptors (Table S2) followed by adding 2 μL 0.5 M EDTA to stop the reaction. Then, these barcoded samples were pooled together and purified using the phenol-chloroform nucleic acid extraction method. The purified nucleic acid pellet was resuspended in TE buffer followed by DRIP and library construction for sequencing as described in ssDRIP-seq.