Pyromark q24 1

Genomic DNA (0.5 μg) was bisulphite-modified using the EZ DNA methylation kit (Zymo Research Corp, Orange County, CA). Polymerase chain reaction (PCR) was carried out with biotinylated forward primers and reverse primers for 21 selected genes (supplementary Table S1), using the following PCR program: 95 °C for 5 min, then 45 cycles of 95 °C for 30 sec followed by 56~61 °C for 30 sec and 72 °C for 30 sec, with a final extension at 72 °C for 5 min. Pyrosequencing was conducted using the PyroMark Q24 1.010 instrument and software (Qiagen). The amount of C relative to the sum of the amounts of C and T at each CpG site was calculated as the percentage of methylated cytosines [6 (link)].
Pyro-sequencing was applied to validate the DNA methylation levels over 26 CpG sites of 21 selected genes identified by the microarray experiment, including RNASE3, MRPS18B, LGALS3, MIR223, ICAM2, GHRL, MIR505, PARP9, PLCL2, WIPI2, PYCR2, ITSN1, ICOS, FOXO3, RPTOR, CCR6, CASP8, GNG12, RASGRP4, GZMK, and MAP1LC3 (Table S4). Primer sequences used in quantitative RT-PCR for the 9 candidate genes are as listed in Table S5.

Genome-wide DNA methylation profiles were measured by Infinium HumanMethylation 450 BeadChip v1.2 microarray method (San Diego, CA, USA). We filter out 485 CpG sites that have bead count smaller than 3 in 5% of total samples and filter out 901 CpG sites that have detection p-value greater than 0.01 in 5% of the total samples with R package wateRmelon^{54 (link)}. We then transfer the methylation β value into M values^{55 (link)} which has better statistical properties for latter non-parametric statistical analysis. For those differentially methylated CpG sites, their corresponding gene symbols were used for pathway analysis and interaction networks contruction by MetaCore software (Thomson Reuters Incorporation, Philadelphia, USA). The significance threshold was a p < 0.0005 and a false discovery rate (q) < 0.3. All methylation datasets have been deposited in the NCBI Gene Expression Omnibus with the accession number GSE118468. Significantly differentially methylated CpG sites with at least a 10% difference in their β value (large effect size) and known biological or functional relevance were selected for further verification and validation by pyrosequencing method using PyroMark Q24 1.010 (Qiagen)^{56 (link)} (Supplementary Appendix 1 Text).

Pyrosequencing was used to validate DNAm at five selected genes that showed differential methylation (P<0.05, FDR corrected) between preterm infants at TEA and term controls: SLC7A5, SLC1A2, NPBWR1 and QPRT. APOL1 was included in validation studies because of its functional relevance and the significance value from the array was marginal (P=0.05). Bisulphite conversion was performed on 500 ng of genomic DNA with the EZ DNAm kit (Zymo Research, Freiburg, Germany). The converted DNA was amplified using the AmpliTaq Gold 360 kit (Applied Biosystems, Warrington, UK) with primers mapping to target regions containing CpGs assayed within the array. PCR primers were designed using PyroMark Assay Design Software 2.0 (Qiagen; https://www.qiagen.com). Pyrosequencing was performed using PyroMark Q24Gold reagents on a PyroMark Q24 Pyrosequencer (Qiagen) according to the manufacturer's instructions. Data were extracted and analysed using PyroMark Q24 1.0.10 software (Qiagen). Background non-conversion levels were ~1–3%.