title: GSM5366058: Senescence of the first seeds_2B; Arabidopsis thaliana; Bisulfite-Seq
library_construction_protocol: The leaves were flash frozen in liquid nitrogen and stored at -80 o C. 6 leaves were pooled together at random to produce 3  replicates per developmental stage. The pooled leaves were ground together in liquid nitrogen to a fine  powder. Approx. 100 mg of plant material were added to 800 µl SLS Buffer + 40 µl Proteinase K  (analytikjena Innu prep plant DNA kit) and incubated at 65 o C for 1h. The samples were loaded on  prefilter (analytikjena Innu prep plant DNA kit) and centrifuged at 11000x g for 1 min. 8 µl of 100 ng  RNAseA / µl were added and incubated at 37 o C for 30 min. DNA was separated with 800 µl  phenol:chloroform:isoamylalcohol (25:24:1) followed by centrifugation for 20 min at 4 o C and max  speed. 700 µl ice cold isopropanol with 70 µl 3M Na Acetate (pH 4.8) was used for DNA precipitation  following incubation at -25 o C for 40 mins. To wash the DNA pellet 750 µl 70% ethanol were added and  incubated at room temperature for 20 mins. Washing with ethanol was repeated 3 times. 50 µl of TE  buffer pH 8 were used to dissolve the DNA pellet. The DNA samples were sent to Novogene Co. Ltd , for WGBS on the Illumina, 150 PE platform with a coverage of 3G (22x). A total amount of 5.2 microgram genomic DNA spiked with 26 ng lambda DNA were fragmented by sonication to 200-400 bp with Covaris S220, followed by end repair and adenylation. Cytosine-methylated barcodes were ligated to sonicated DNA as per manufacturer's instructions. The DNA fragments were then treated twice with bisulfite using EZ DNA Methylation- Gold TM Kit (Zymo Research). The resulting single-strand DNA fragments were PCR amplificated using KAPA HiFi HotStart Uracil + ReadyMix (2X). Library concentration was quantified by Qubit® 2.0 Flurometer (Life Technologies, CA, USA) and quantitative PCR, and the insert size was checked on Agilent 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using PE Cluster Kit cBot-HS (Illumina) according to the manufacturer's instructions.