title: GSM2200170: Sample 5_LUCH_r2 MethylC-seq; Arabidopsis thaliana; Bisulfite-Seq
library_construction_protocol: For the MethylC-seq libraries corresponding to the “_r2, _r5, and_B” series (Table S4), genomic DNA was extracted using the DNeasy Plant Mini  Kit (Qiagen, Cat# 69104). One microgram of genomic DNA was sonicated into  fragments 150 to 300 bp in length using a Diagenode Bioruptor, followed by purification with the PureLink PCR Purification Kit (Invitrogen, Cat#  K3100-01). For all the remaining MethylC-seq libraries (Table S4), genomic  DNA was extracted from 10-day-old seedlings using the DNeasy Plant Mini Kit  (Qiagen, Cat# 69104). 2 ug of genomic DNA was sonicated into 200 bp fragments using a Covaris S2 Sonicator, followed by purification with Sera-Mag Magnetic SpeedBeads (ThermoScientific, Cat# 65152105050250)." For the MethylC-seq libraries corresponding to the '_r2, _r5, and_B' series  (Table S4), the sonicated and purified genomic DNA fragments were repaired using the End-It DNA End-Repair Kit (Epicentre, Cat# ER0720), and the DNA fragments were purified using the Agencourt AMPure XP-PCR Purification system (Beckman Coulter, Cat# A63880). The purified DNAs were adenylated at the 3? end using the polymerase activity of Klenow Fragment (3?"5? exo-) (New England Biolabs, Cat# M0212), followed by purification using the Agencourt AMPure XP-PCR Purification system. The methylated adapters in the TruSeq DNA Sample Preparation Kit (Illumina, Cat# FC-121-2001) were ligated to the DNA fragments using T4 DNA Ligase (New England Biolabs, Cat# M0202). After purification using AMPure XP beads, less than 400 ng DNA was subjected to bisulfite conversion using the MethylCode Bisulfite Conversion Kit (Invitrogen, Cat# MECOV-50). PfuTurbo Cx Hotstart DNA polymerase (Agilent, Cat# 600414) and the following PCR conditions were used for amplification: 95°C for 2 minutes; 9 cycles of 98°C for 15 seconds, 60°C for 30 seconds and 72°C for 4 minutes; and 72°C for 10 minutes. The resulting libraries were quantified, pooled and sequenced using HiSeq 2000 with the 101-cycle single-end sequencing mode (Illumina). For all the remaining MethylC-seq libraries (Table S4), the sonicated and purified genomic DNA fragments were repaired using the End-It DNA End-It DNA End-Repair Kit (Epicentre, Cat# ER81050), and the DNA fragments were purified using Sera-Mag Magnetic SpeedBeads. The purified DNAs were adenylated at the 3? end using the polymerase activity of Klenow Fragment (3?"5? exo-) (New England Biolabs, Cat# M0212), followed by purification using Sera-Mag Magnetic SpeedBeads. The methylated adapters in the NEXTflex Bisulfite-Seq Barcodes Kit (BIOO Scientific, Cat# 511911) were ligated to the DNA fragments using T4 DNA Ligase (New England Biolabs, Cat# M0202). After purification using Sera-Mag Magnetic SpeedBeads, the DNA was subjected to bisulfite conversion using the MethylCode Bisulfite Conversion Kit (Invitrogen, Cat# MECOV-50). KAPA HiFi HotStart Uracil+ ReadyMix PCR Kit (KAPA Biosystems, Cat# KK2801) and the following PCR conditions were used for amplification: 95°C for 2 minutes; 98°C for 30 seconds; 4 cycles of 98°C for 15 seconds, 60°C for 30 seconds and 72°C for 4 minutes; and 72°C for 10 minutes. After purification using Sera-Mag Magnetic SpeedBeads, the libraries were quantified, pooled and sequenced at single-end 50 base-pair using the Illumina HiSeq 2500 v4 at the Salk NGS Core. Raw sequencing data was de-multiplexed and converted into FASTQ files using CASAVA (v1.8.2)
attributes.tag: GEO Accession
attributes.value: GSM2200170