Total RNA was isolated from 10-20 mg of endometrium and thymus of each fetus using an ‘All Prep RNA/DNA isolation Mini kit’ (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA was quantified by spectrophotometry using a Nanodrop ND 2000 (Thermo Fisher Scientific, Waltham, USA). RNA quality was assessed by digital electrophoresis using a ‘2200 Tapestation’ (Agilent Technologies, Santa Clara, USA). The mean RNA Integrity Number (RIN) sample value was 9.0 for thymus (range 8.0-9.8) and 8.3 for endometrium (range 7.5-8.8). A total of 96 cDNA libraries were constructed for RNA-sequencing (RNA-seq) from thymus and endometrium samples of individual fetuses. Libraries were constructed from 1 μg of input total RNA using the TruSeq RNA sample preparation kit v2 (Illumina, San Diego, USA) according to the manufacturer’s instructions. DNA libraries were sequenced at the Genome Quebec Innovation Centre (McGill University, Montreal, Canada). Library insert size distribution was assessed on a LabChip GX instrument (Perkin Elmer, Waltham, USA) using a high sensitivity chip. Library concentration was determined using a KAPA Library Quantification Kit (KAPA Biosystems) on a Lightcycler 480 II instrument (Roche Life Science, Indianapolis, USA). Cluster formation was performed on the cBot instrument (Illumina) with eight libraries per lane (7 pM per library) using the TruSeq PE Cluster Kit v3-cBot-HS (Illumina). Paired-end 100 bp cycle sequencing was performed on a HiSeq 2000 (Illumina) using the TruSeq SBS kit v3-HS (Illumina).
GEO Accession
GSM1829839