GSM2977093: Tissue from nonADPKD kidney 2; Homo sapiens; Bisulfite-Seq
Genomic DNA was extracted from fresh-frozen sections of kidney tissue using the QIAmp DNA Mini kit (Qiagen, Hilden, Germany) following the manufacturer's protocol. Libraries prepared from Kidney E1, G2, H3, ADPKD 07 and 08 were prepared from 2.5µg of DNA. Due to improvements in the methodology, the subsequent preparation of libraries from ADPKD 05 and D only required an input of 500ng of DNA, and did not require size selection before bisulfite conversion. Genomic DNA was digested with MspI (New England Biolabs, Ipswich, MA), followed by end repair and the addition of 30 A overhangs. Methylated adaptors (Illumina, San Diego, CA) with a 30 T overhand were then ligated to the generated fragments. Following adaptor ligation, fragments ranging from 40 to 220 bp (preligation size) were cut from a 3% (w/v) NuSieve GTG Agarose gel (Lonza, Basel, Switzerland), and were subsequently bisulfite modified using the EZ DNA Methylation kit (Zymo Research, Irvine, CA) (ADPKD D was converted with the MethylCode Bisulfite Conversion kit (Invitrogen, Carlsbad, CA)) before selecting fragments between 40 and 220bp from NuSieve gel. This library was amplified by PCR, and the resulting library was sequenced on an Illumina platform with a single-ended 100bp run.