RRBS investigation of matched side-population trophoblast, cytotrophoblast and extravillous trophoblast populations from 4 human placentae

To better understand how DNA methylation influences placentation, DNA from first trimester primary trophoblast populations (side-population trophoblasts, cytotrophoblasts and extravillous trophoblasts) isolated using FACS underwent reduced representation bisulfite sequencing and were compared to publicly available data of blastocyst-derived and somatic cell populations. Overall design: Comparisons were made between three matched trophoblast populations (side-population trophoblasts, cytotrophoblasts, extravillous trophoblasts) from four first trimester human placentae isolated using FACS. DNA from each of the three trophoblast populationswas extracted using a Qiagen DNA Mini Kit (Qiagen, Germany). DNA quantity was assessed using a Qubit® dsDNA HS Kit (Molecular Probes, USA) or Qubit® dsDNA BR Kit (Molecular Probes, USA). RRBS was undertaken on 500ng of DNA per sample as previously described (Chatterjee et al., 2012a) using the MspI restriction enzyme in the RRBS library preparation with one size selection step (150-325bp). Libraries were amplified with 15–18 cycles. RRBS libraries underwent single-ended (100bp) sequencing using an Illumina HiSeq2000 (Chatterjee et al., 2012b). The reads were aligned to human GRCh37 reference genome assembly using Bismark aligner (Krueger and Andrews, 2011). The resulting bam files were sorted and Bismark methylation extractor (Krueger and Andrews, 2011) was used to determine DNA methylation status and to yield CpG report files. Publicly avaliable data of blastocyst-derived and somatic cell populations were obtained from SRA for comparison. Analysis of these report files was performed using R package “methylKit” (Akalin et al., 2012).