Epitaq hs

Genomic DNA was isolated from TGS‐01 and TGS‐04 cells, and bisulfite treatment was performed using an EpiTect Bisulfite Conversion Kit (Qiagen). Using the bisulfite‐treated genomic DNA as a template, PCR was carried out using EpiTaq HS (Takara Bio). The PCR products were analyzed by sequencing or agarose gel electrophoresis. The primers used for the PCR are listed in Tables S6 and S7.

Genomic DNA was extracted a using Genomic DNA Extraction Kit Ver.5.0 (TaKaRa Code. 9765). Bisulphite sequencing aliquots of 800 ng DNA were treated with sodium bisulphite using the EpiTect Fast Bisulfite Kit (QIAGEN, Cat. 59824, city, state if US, country,) according to the manufacturer’s instructions. DNA was amplified by PCR with EpiTaq™ HS (Takara Code No. R110). PCR products were cloned into the pMD19-T Simple Vector (Takara Code No. 3271) and the clones were sequenced. The amplification parameters were as follows: 95 °C for 5 min, 40 cycles of (94 °C for 30 s, 542 °C for 30 s, 72 °C for 40 s), and 72 °C for 5 min. For each region, more than 20 independent top-strand clones for were sequenced from each sample.

Mouse alveolar lung tissues attached to LCM caps were stored at −80°C until processing. DNA was extracted using the ZR Genomic DNA-Tissue MicroPrep kit (Zymo Research). Incubation with Digestion buffer and proteinase K was done overnight at 55°C in inverted tubes. 13 genes were chosen for targeted NextGen bisulfite sequencing (NGBS): Igfbp3, Wif1, Cdh11, Eln, Sox9, Tnc, Dnmt3a, Akt, Vegfa, Lox, Foxf2, Zfp536 and Src, based on published data (Cuna et al., 2015 (link)). Targeted NGBS was done on samples collected at: E16.5, E18.5, P0.5, P1.5, P2.5, P5, P10, P15, P19 and P26. Multiplex PCR was performed using 0.5 units of TaKaRa EpiTaq HS (Takara Bio, Kusatsu, Japan) in 2x master mix. FASTQ files were aligned using open source Bismark Bisulfite Read Mapper using Bowtie2. Methylation levels were calculated in Bismark. Sites where the difference in methylation was less than 5% over the entire time period, those where there was a difference of >20% at a single time point and those with less than $3$ non zero values were removed from the analyses.

Genomic DNA was extracted from cells with the DNeasy Blood & Tissue Kit (Qiagen). Bisulphite modification of DNA was performed with the EZ DNA Methylation-Gold Kit (Zymo Research) according to the manufacturer’s recommendations. Regions within the KvDMR or the Cdkn1c sDMR were PCR amplified with TaKaRa EpiTaq HS (Takara), using primers that specifically recognize bisulphite-converted DNA (sequences are provided in Table 1). PCR products were separated by agarose gel electrophoresis and bands corresponding to the predicted size were excised and purified with a QIAquick Gel Extraction Kit (Qiagen). Purified products were ligated into the pJET1.2/blunt cloning vector using the CloneJET PCR Cloning Kit (Thermo Scientific), as per the manufacturer’s protocol, before heat-shock transformation into DH5α competent cells. Bacteria were plated onto LB/ampicillin plates and grown overnight at 37 °C. Colonies were picked (18–24 per sample) and expanded in 1 ml LB/ampicillin broth overnight at 37 °C. The following morning plasmids were purified with the Wizard SV 96 Plasmid DNA Purification System (Promega), according to the manufacturer’s protocol, and sent for Sanger sequencing with the PJET1-2R primer (GENEWIZ). Sequences with < 90% conversion of non-CpG Cs were excluded.