GSM2088144: LC-T-5; Homo sapiens; Bisulfite-Seq
Genomic DNAs were extracted from 20-50 mg of primary normal and tumor tissues from lung, colon and pancreatic cancer patients using DNeasy Blood and Tissue kit (QIAGEN) following the manufacturer’s instruction and eluted in 400 µL of AE buffer (QIAGEN). The concentration and quality of genomic DNA were assessed by Qubit dsDNA HS Assay kit (Life Technologies) and NanoDrop (Thermo Scientific), respectively. To generate RRBS sequencing libraries, 100 ng of gDNA were digested with 20 U of MspI (Thermoscientific) in 1X Tango buffer (Thermoscientific) and 1 ng of unmethylated lambda DNA (Promega) in order to assess for bisulfite conversion rate in 30µL total volume for 3 h at 37 ˚C and heat inactivated at 65 ˚C for 20 min. Next, 5U of Klenow fragment, exo- (Thermoscientifc) and a mixture of dATP, dGTP, and dCTP (New England Biolabs) were added to MspI-digested DNAs for a final concentration of 1 mM, 0.1 mM, and 0.1 mM for dATP, dGTP, and dCTP, respectively in 32 µL for end-repair and dA-tailing. The mixture was mixed and incubated at 30 ˚C for 20 min, 37 ˚C for 20 min, and heat inactivated at 75 ˚C for 10 min. dA-tailed DNA was purified with 2X volume of Agencourt AMPure XP beads (Beckman Coulter) and resuspend dA-tailed DNA with 20 µL nuclease-free water without discarding the magnetic beads. dA-tailed DNAs were then ligated to methylated adaptors in 30 µL total volume containing 30 U of T4 DNA ligase, HC (Thermoscientific), 1X Ligation buffer (Thermoscientific), and 500 nM individual TruSeq multiplexing methylated adaptors (Illumina). The ligation mixture was mixed well and incubated at 16 ˚C for 20 h, heat inactivated at 65 ˚C for 20 min, purified by adding 60 µL of PEG 8000/5M NaCl buffer (Teknova) to adaptor ligated DNA and bead mixture, and eluted in 20 µL of nuclease-free water. Next, the adaptor ligated DNA were bisulfite converted using the MethylCode(™) Bisulfite Conversion kit (Life Technologies) following manufacturer’s protocol and eluted in 35 µL of Elution buffer (Life Technologies). Bisulfite treated DNAs were amplified using 5 U of PfuTurboCX (Agilent Technologies) and 300 nM each of TruS_F and TruS_R primers for 14 cycles in 100 µL total volume. PCR products were purified with an equal volume of Agencourt AMPure XP beads (Beckman Coulter) and eluted with 50 µL of 10mM Tris-HCl, pH8.5, pooled in equimolar ratios, and size selected using 6% TBE gels for 150-400 bp. The concentration of sequencing libraries was quantified by qPCR using KAPA Library Quantification kit (KAPA Biosystems). Libraries were sequenced on HiSeq RapidRun for PE 100 cycles.
GEO Accession
GSM2088144