alias: GSM3409496
title: GSM3409496: Tcast_2: Tribolium castaneum methylation 2; Tribolium castaneum; Bisulfite-Seq
library_construction_protocol: Embryos were dechorionated in diluted sodium hypochlorite bleach, washed and then smashed in SDS buffer using an Eppendorf micropestle. After 30 min incubation at 65 °C and RNAse digestion, remaining RNAs were precipitated with LiCl/KAc. After centrifugation, the supernatant was taken and DNA was precipitated with isopropanol, washed in 70% ethanol and then resuspended in TE. Per sample 5 µg of high molecular weight DNA were used for fragmentation using the Covaris S2 AFA System in a total volume of 100 µl. The fragmentation (parameters: duty cycle: 10 %; intensity: 5; cycles/burst: 200; cycles: 3) was run for a total fragmentation time of 180 s. Fragmentation was confirmed with a 2100 Bioanalyzer (Agilent Technologies) using a DNA1000 chip. The fragmented DNAs were concentrated to a final volume of 75 µl using a DNA Speed Vac. End repair of fragmented DNA was carried out in a total volume of 100 µl using the Paired End DNA Sample Prep Kit (Illumina) as recommended by the manufacturer. For the ligation of the adaptors, the Illumina Early Access Methylation Adaptor Oligo Kit and the Paired End DNA Sample Prep Kit (Illumina) were used, as recommended by the manufacturer. For the size selection of the adaptor-ligated fragments, we used the E-Gel Electrophoresis System (Invitrogen) and a Size Select 2 % precast agarose gel (Invitrogen). Each fragmented DNA was loaded on two lanes of the E-gel. Electrophoresis was carried out using the “Size Select” program for 16 min. According to the standard loaded (50 bp DNA Ladder, Invitrogen), 240 bp fragments were extracted from the gel, pooled, and directly transferred to bisulfite treatment without further purification. For the bisulfite treatment we used the EZ-DNA Methylation Kit (Zymo) as recommended by the manufacturer with the exception of a modified thermal profile for the bisulfite conversion reaction. The conversion was carried out in a thermal cycler using the following thermal profile: 95 °C for 15 s, 50 °C for 1 h, both steps repeated 15 times, 4 °C for at least 10 min. The libraries were subsequently amplified, using the Fast Start High Fidelity PCR System (Roche) with buffer 2, and PE1.1 and PE2.1 amplification primers (Illumina). PCR thermal profile: 95 °C for 2 min, 95 °C for 30 s, 65 °C for 20 s, 72 °C for 30 s, then repeat from step 2, 11×, 72 °C for 7 min, hold at 4 °C. PCR reactions were purified on PCR purification columns (MinElute, Qiagen) and eluted in 20 µl elution buffer (Qiagen).
identifiers.namespace: GEO
identifiers.id: GSM3409496
attributes.tag: GEO Accession
attributes.value: GSM3409496