title: GSM1908381: Carp_4_G; Cyprinus carpio; Bisulfite-Seq
library_construction_protocol: The Allprep DNA/RNA Mini kit (Qiagen) was used for DNA isolation. Cells were lysed in 600 µl Buffer RLT Plus supplemented with 1% β-Mercaptoethanol and vortexed thoroughly for at least five minutes. The procedure of isolating DNA and RNA was performed according to protocol. DNA was stored at -20˚C. For RRBS, 100 ng of genomic DNA was digested for 12 hours at 37°C with 20 units of MspI (New England Biolabs, R0106L) in 30 μl of 1x NEB buffer 2. To retain even the smallest fragments and to minimize the loss of material, end preparation and adaptor ligation were performed in a single-tube setup. End fill-in and A-tailing were performed by addition of Klenow Fragment 3’ > 5’ exo- (New England Biolabs, M0212L) and dNTP mix (10 mM dATP, 1 mM dCTP, 1 mM dGTP). After ligation to methylated Illumina TruSeq LT v2 adaptors using Quick Ligase (New England Biolabs, M2200L), the libraries were size selected by performing a 0.75x clean-up with AMPure XP beads (Beckman Coulter, A63881). The libraries were pooled in combinations of six based on qPCR data and subjected to bisulfite conversion using the EZ DNA Methylation Direct Kit (Zymo Research, D5020) with the following changes to the manufacturer’s protocol: conversion reagent was used at 0.9x concentration, incubation performed for 20 cycles of 1 min at 95°C, 10 min at 60°C and the desulphonation time was extended to 30 min. These changes increase the number of CpG dinucleotides covered, by reducing double-strand break formation in larger library fragments. Bisulfite-converted libraries were enriched using PfuTurbo Cx Hotstart DNA Polymerase (Agilent, 600412). The minimum number of enrichment cycles was estimated based on a qPCR experiment. After a 2x AMPure XP clean-up, quality control was performed by a Qubit dsDNA HS (Life Technologies, Q32854) and Experion DNA 1k assay (BioRad, 700-7107). The RRBS libraries were sequenced on the Illumina HiSeq 2000 platform in 50 bp single read mode.
attributes.tag: GEO Accession
attributes.value: GSM1908381