GSM3358373: wtJ1_1; Mus musculus; Bisulfite-Seq
Genomic DNA was isolated from 2x106 cells using the PureLink Genomic DNA Mini Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. The EZ DNA Methylation-Gold Kit (Zymo Research) was used for bisulfite conversion according to the manufacturer's instructions with 500 ng of genomic DNA used as input and the modification that bisulfite converted DNA was eluted in 2 x 20 µL Elution Buffer. The sequences of the LINE1 specific primers were appended with Illumina TruSeq and Nextera compatible overhangs. The amplification of bisulfite converted DNA was performed in 25 µL PCR reaction volumes containing 0.4 µM each of forward and reverse primers, 2 mM Betaine (Sigma-Aldrich, B0300-1VL), 10 mM Tetramethylammonium chloride solution (Sigma-Aldrich T3411-500ML), 1x MyTaq Reaction Buffer, 0.5 units of MyTaq HS (Bioline, BIO-21112), and 1 µL of the eluted bisulfite converted DNA (~12.5 ng). The following cycling parameters were used: 5 min for 95°C for initial denaturation and activation of the polymerase, 40 cycles (95 °C for 20 s, 58°C for 30 s, 72°C for 25 s) and a final elongation at 72°C for 3 min. Agarose gel electrophoresis was used to determine the quality and yield of the PCR. For purifying amplicon DNA, PCR reactions were incubated with 1.8x volume of CleanPCR beads (CleanNA, CPCR-0005) for 10 min. Beads were immobilized on a DynaMag™-96 Side Magnet (Thermo Fisher, 12331D) for 5 min, the supernatant was removed, and the beads washed 2x with 150 µL 70% ethanol. After air drying the beads for 5 min, DNA was eluted in 15 µL of 10 mM Tris-HCl pH 8.0. Amplicon DNA concentration was determined using the Quant-iT™ PicoGreen™ dsDNA Assay Kit (Thermo Fisher, P7589) and then diluted to 0.7 ng/µL. Thereafter, indexing PCRs were performed in 25 µL PCR reaction volumes containing 0.08 µM (1 µL of a 2 µM stock) each of i5 and i7 Indexing Primers, 1x MyTaq Reaction Buffer, 0.5 units of MyTaq HS (Bioline, BIO-21112), and 1 µL of purified PCR product (diluted to 0.5 ng/µl) from the previous step. The following cycling parameters were used for the indexing PCR: 5 min for 95°C for initial denaturation and activation of the polymerase, 15 cycles (95°C for 10 s, 55°C for 30 s, 72°C for 40 s) and a final elongation at 72°C for 5 min. Agarose gel electrophoresis was used to determine the quality and yield of the PCR. An aliquot from each indexing reaction (5 µL of each reaction) was then pooled and purified with CleanPCR magnetic beads as described above and eluted in 1 µL x Number of pooled reactions. Concentration of the final library was determined using PicoGreen and the quality and size distribution of the library was assessed with a Bioanalyzer. Targeted bisulfite amplicon sequencing
GEO
GSM3358373