GSM9323458: LTL331RPDXcombo_dAZA_3 (eRRBS); Homo sapiens; Bisulfite-Seq
Total DNA was extracted using a Qiagen AllPrep RNA/DNA mini kit (Qiagen 80204) according to the manufacturer's protocol. 25-75 ng of genomic DNA was digested for 16-18 hours with the MspI restriction enzyme. The digested DNA was purified by phenol-chloroform extraction, then ethanol precipitated in the presence of glycogen and sodium acetate. The precipitated DNA was resuspended in 10mM Tris-Cl pH8.5 (Buffer EB) and processed for end-repair, A-tailing, and ligation of hairpin adapters with methylated cytosines. The ligated DNA was next ran on a 1.8% Low-Range Ultra Agarose Gel in TBE buffer, and fragments of 100-200bp and 200-300bp were excised and separately purified using Qiagen Gel Extraction kit. A 2ul aliquot of each fragment was used to verify proper adapter ligation and size selection by PCR. Samples passing this QC were then subjected to bisulfite conversion with the EZ DNA Methylation Kit, but with the following changes: the samples were incubated in a thermal cycler using the following conditions: 55 cycles: 95C for 30 sec, 50C for 15 min. Hold at 4C. The bisulfite converted samples were cleaned up according to the manufacturer's protocol and eluted in 40ul of nuclease free water. The samples were next amplified using Roche High Fidelity FastStart system with the following protocol: 94C for 5 minutes; 18 cycles of: denaturing at 94C for 20 seconds, annealing at 60C for 30 seconds, extension at 72C for 1 min. A final extension was done at 72C for 3 minutes and the reactions were kept at 4C until they were cleaned. This final library cleanup was done with Agencourt's AMPure XP SPRI beads according to the following protocol: The beads were brought to room temperature with gentle agitation for a minimum of 30 minutes. A 1.2X volume (for short fragments) and 1.1X volume (for long fragments) of SPRI beads was added to each sample fragment, and the PCR products were purified per manufacturer's recommendation using 500ul of 80% ethanol for washes. Each library was eluted into 33ul of elution buffer (10mM Tris-Cl pH 8.5). The libraries were quantified using Qubit's High Sensitivity ds DNA kit, and the quality was assessed with Agilent's High Sensitivity D1000 ScreenTape assay. Libraries for Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) were prepared according to (Garrett-Bakelman et al., 2015)
GEO
GSM9323458_r1