title: GSM1195537: Male#1 Brain; Mus musculus; Bisulfite-Seq
library_construction_protocol: Tissues were harvested and lysed using tail lysis buffer containing Proteinase K.  Genomic DNA was isolated and purified using the phenol–chloroform extraction method (Sigma-Aldrich, Cat# P2069). Briefly, 1µg of the isolated genomic DNA was sonicated to fragments with their average size being 700 bp in length (Bioruptor NGS, Diagenode).  Since the average lengths of IAPLTR are 300-350 bp in length and the desired sequences need to contain non-repeat flanking sequences, the optimum size of the fragments are empirically determined to be around 700 bp in length.  The fragmented DNA was immediately end-repaired using the NEB Next End Repair Module (New England BioLabs), and then ligated to custom-made duplex Ion Torrent ‘A’ adaptors using T4 DNA ligase (New England BioLabs).  In these adaptors, all the Cs have been methylated (Integrated DNA Technologies).  The adaptor-ligated DNA fragments were size-selected to remove any fragment smaller than 300 bp in length using the Agencourt AMPure XP beads (Beckman Coulter).  The size and quantity of the selected fragments were analyzed using the Agilent 2100 Bioanalyzer.  The adaptor-ligated DNA library was modified using the bisulfite conversion reaction according to the manufacturer’s protocol (EZ DNA methylation kit, Zymo Research).  The bisulfite-converted ‘A’ adaptor-ligated library was used as a template for a round of PCR (Maxime PCR Premix Kit, Intron Biotech).  In this PCR, the forward primer (CCATCTCATCCCTGCGTGTCTCCGACTCAG) was designed to bind to the 5’-end of the ‘A’ adaptor region whereas the reverse primer was designed to bind to the 24-bp conserved region among the consensus sequences of the IAPLTR subtypes (IAPLTR1, IAPLTR1a, IAPLTR2, IAPLTR2a, and IAPLTR2b).  The 5’-end of the reverse primer also had the sequence of the regular Ion Torrent ‘P1’ adaptor (CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT^CTCCCTAATTAACTACAACCCATC).  The break in the reverse primer sequence indicates the joining point of the P1 adaptor sequence and the IAPLTR-specific sequence.  The amplification cycle number of PCR was individually determined for each of the twelve samples so that the minimum possible cycles were used to generate just enough amount of the PCR product for sequencing.  The PCR product was size-selected for a range of 250-300 bp in length using agarose gel electrophoresis.  The size-selected PCR product was verified for its length and quantity using the Agilent 2100 Bioanalyzer.  Finally, each of the twelve PCR products was individually sequenced in the Ion Personal Genome Machine (PGM) Sequencer using Ion 318 Chips (Ion Torrent, Life Technologies).
attributes.tag: GEO Accession
attributes.value: GSM1195537