title: GSM6722278: NIP_rep2; Oryza sativa; RNA-Seq
library_construction_protocol: sRNA-seq: total RNA was purified from 4-week-old seedlings using TRIzol reagent (Invitrogen, 15596026). Large RNAs were selectively precipitated using 5% polyethylene glycol 8000 (PEG8000; Sigma, P5413) and 0.5 M NaCl (Sigma, S3014). sRNAs were recovered from the supernatant by ethanol precipitation and then separated by electrophoresis using a 15% denaturing PAGE gel. sRNAs that were 18–28 nt long were excised from the gel and purified.  WGBS-seq: genomic DNA was purified from the 4-week-old seedlings using the DNeasy Plant Mini Kit (Qiagen, 69104). sRNA-seq: purified sRNAs were ligated with the 3' adaptors and 5' adaptors. The products were reversely transcribed using SuperScript III Reverse Transcriptase (Invitrogen, 18080093). The cDNAs obtained were amplified by PCR to generate the libraries. The libraries were sequenced on an Illumina HiSeq2500 platform to obtain 50-bp single end reads.   WGBS-seq: Paired-end bisulfite sequencing libraries were constructed using the NEXTflex Bisulfite-Seq kit (Bioo Scientific, Nova-511911). Genomic DNA was sonicated to ~300 bp, end repaired, and 3' adenylated. The products were ligated with methylated adapters. After size selection with Ampure XP purification beads (Beckman, A63881), the products were subjected to bisulfite conversion by using the EZ DNA Methylation-Gold kit (ZYMO Research, D5005). 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 libraries were validated using the Agilent 2100 Bioanalyzer and sequenced on an Illumina X-Ten platform to obtain 150-bp paired-end reads.