GSM3769096: osnrpd1ab_base_BSseq180209_rep3; Oryza sativa Japonica Group; Bisulfite-Seq
For whole-genome bisulfite sequencing, genomic DNA was purified from the shoot base of 4-week-old seedlings using the DNeasy Plant Mini Kit (QIAGEN, 69104). For RNA sequencing, total RNA was purified from the shoot base of 4-week-old seedlings using the RNeasy Plant Mini Kit (QIAGEN, 74904). For small RNA sequencing, total RNA was purified from the shoot base of 4-week-old seedlings using TRIzol reagent (Invitrogen, 15596026). Large RNAs were selectively precipitated using 5% PEG8000 (Sigma, P5413) and 0.5 M NaCl (Sigma, S3014). Small RNAs were recovered from the supernatant by ethanol precipitation and then separated by electrophoresis using a 15% denaturing PAGE gel. Paired-end bisulfite sequencing libraries were generated using the NEXTflex Bisulfite-Seq kit (BIOO SCIENTIFIC, NOVA-511911). Briefly, genomic DNA was sonicated to ~300 bp, end repaired and 3' adenylated. The products were ligated with methylated adapters and then subject to size selection using Ampure XP purification beads (Beckman, A63881). Bisulfite treatment was carried out using the EZ DNA Methylation-Gold kit (ZYMO Research, D5005) according to the manufacturer's instructions. Bisulfite-converted DNA products were amplified by PCR for 15 cycles using EX Taq DNA polymerase (Takara, RR001C). The PCR products were cleaned up using Ampure XP purification beads (Beckman, A63881). The constructed libraries were validated using the Agilent 2100 Bioanalyzer and paired-end sequenced in 150-bp on an Illumina X-Ten platform. Libraries for RNA sequencing were generated using NEXTflex RNA-Seq Kit (Bioo Scientific) according to the manufacturer's instructions. Libraries for small RNA sequencing were generated using NEBNext Small RNA Library Prep Set for Illumina (NEB).
GEO Accession
GSM3769096