title: GSM3834690: dme-2_DMEFL-1_BS-seq_endo; Arabidopsis thaliana; Bisulfite-Seq
library_construction_protocol: Genomic DNA were isolated from hand dissected, 5-6 DAP (torpedo to early-bend stage seed stage) dme-2 endosperm that has been complemented by nDMECTD (dme-2/dme-2; nDMECTD/nDMECTD). Approximately 20-50 ng of purified genomic DNA was sheared to about 300bp using Covaris M220 (Covaris Inc., Woburn, Massachusetts). The sheared DNA was cleaned up and recovered by 1.2x AMPure XP beads then followed by end repaired and A-tailing (NEBNext Ultra II DNA Library Prep Kit for Illumina, NEB) before ligated to the NEBNext methylated multiplex adaptors (NEBNext Multiplex Oligos for Illumina, NEB) according to the manufacturer's instructions. Adaptor-ligated DNA was cleaned up with 1x AMPure XP beads. The purified adaptor-ligated DNA was subjected to one round of sodium bisulfite conversion using the EZ DNA Methylation-Lightning Kit (Zymo Research Corporation, Irvine, CA) as outlined in the manufacturer's instruction with 80 min of conversion time. Half of the bisulfite-converted DNA molecules was PCR amplified with the following condition: 2.5 U of ExTaq DNA polymerase (Takara), 5 ul of 10 x Extaq reaction buffer, 25 µM dNTPs, 1 ul of universal and index primers (10 uM) in 50 uL reaction. The thermocyling condition was as follows: 95 °C for 2 min and then 10 cycles each of 95 °C for 30 s, 65 °C for 30 s, and 72 °C for 60 s. The enriched libraries were purified twice with 0.8x (v/v) AMPure XP beads to remove adaptor dimers. High throughput sequencing was performed by Novogene Corporation (USA).
attributes.tag: GEO Accession
attributes.value: GSM3834690