GSM4399800: Spleen_1_d34; Gallus gallus; Bisulfite-Seq
For epithelial cell preparation from colon, colon was flushed with ice-cold PBS, cut open longitudinally. Distal and proximal colon region were processed separately. Each part was minced into small pieces in cold PBS. Enterocytes were mechanically isolated by shaking in PBS with 30 mM EDTA, at 37oC. Crypts were recovered and then stored at -80°C for DNA extraction. Only samples from distal colon were used in our study. 5 µg of high molecular weight DNA were used for fragmentation using the Covaris S2 AFA System in a total volume of 100 µl. Fragmentation-run parameters: Duty cycle 10%; Intensity: 5; Cycles/burst: 200; Time: 3 min; number of cycles: 3, resulting in 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, repeat from step 1, 15×, 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 Illuminas PE1.1 and PE2.1 amplification primers. 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 7min, hold at 4°C. PCR reactions were purified on PCR purification columns (MinElute, Qiagen) and eluted in 20 µl elution buffer (Qiagen).
GEO Accession
GSM4399800