title: GSM7350073: 1619_LUNG MOUSE 5 lane7651_TGTGGTTG_1619_LUNG; Mus musculus; OTHER
library_construction_protocol: Genomic DNA was isolated using the Nucleo Spin TriPrep kit (Macherey-Nagel GmbH & Co.KG, Germany) according to manufacturer's instructions. 1μg of genomic DNA was bisulfite converted using the EZ DNA methylation Gold kit (Zymo Research) according to manufacturer's instructions with either magnetic bead or column clean-up and eluted/resuspended in 66μl elution buffer to obtain a final concentration of ^~15ng/μl bisulfite converted DNA. The IMPLICON protocol consists of 2 PCR reactions. In the first reaction each sample is amplified with each primer pair in individual reactions: 30ng (2μl of 66μl eluted bisulfite treated DNA) of bisulfite treated DNA is amplified with 1.2μl of 10μM primer pool (final 1.5μM) containing both forward and reverse primers in 4μl of 2x KAPA HiFi Uracil+ ReadyMix in a final volume of 8μl. DNA was amplified using the following conditions: initial denaturation at 95°C for 5min, 30 cycles of 98°C denaturation for 20 seconds, variable annealing temperature for 15 seconds and extension at 72°C for 60 seconds; followed by a final extension at 72°C for 10 minutes. All PCR reactions for each individual sample were pooled together and samples cleaned-up using 1.5x AMPure XP beads and eluted in 20μl H2O. In the second PCR reaction, barcoded Illumina adapters are attached to the pooled PCR samples ensuring that each sample pool receives a unique reverse barcoded adapter. The 20μl PCR pool was amplified using 1μl of 10μM Illlumina PE1.0 primer (same for all samples) and 1μl of 10μM Illumina iTAG primer (distinct for each sample) and 25μl 2x KAPA HiFi Uracil+ ReadyMix in a 50μl volume reaction using the following conditions: initial denaturation at 98°C for 45 seconds, 5 cycles of 98°C denaturation for 15 seconds, 65°C annealing for 30 seconds and extension at 72°C for 30 seconds; followed by a final extension at 72°C for 5 minutes. Reactions were cleaned-up with 1x AMPure XP beads and eluted in 20μl. Libraries were verified by running 1:30 dilutions on Agilent bioanalyzer. Libraries weere sequenced on an Illumina MiSeq using a 2x250 kit. IMPLICON-seq