GSM9258278: MethylCseq_Anthers_pol-iv; Arabidopsis thaliana; Bisulfite-Seq
Genomic DNA was extracted from anthers (stage 9) and ovules (stage 12) using the CTAB method followed by RNA digestion with RNase A (Thermo, #EN0531) according to the manufacturer's instructions. 2 μg of genomic DNA was fragmented using a Covaris S2 sonicator. The sonicated DNA was size-selected using SpeedBeads (Thermo Scientific #65152105050250) to collect fragments 150-300 bp in length. The DNA ends were repaired using the End-It DNA repair kit (epicenter # ER81050) and purified using SpeedBeads. The end-repaired fragments were adenylated using the polymerase activity of the Klenow Fragment (N-terminal truncation of DNA Polymerase I, NEB # M0212L), purified using SpeedBeads, and indexed using Trueseq Adapters (BIOO Scientific # 511911) and T4 DNA ligase (NEB # M0202). After purification using SpeedBeads, the fragments were subjected to bisulfite conversion using the MethylCode Bisulfite Conversion Kit (Invitrogen, MECOV-50). Bisulfite-converted DNA was amplified by PCR using the KAPA HiFi HotStart DNA polymerase (KAPA BIOSYSTEMS # KK2801). The following program was used for the PCR amplification: 95˚C for 2 min; 98˚C for 30 s; 4 cycles of (98˚C for 15 s, 60˚C for 30 s, 72˚C for 4 min); and 72˚C for 10 minutes. The PCR product was purified using SpeedBeads and pooled for sequencing using the pair-end 50 bp kit (PE50) on the Illumina Nextseq 2000 or Novaseq X plus platform.