Agilent 6000 nano chip

Total RNA was isolated using the mirVanaTM miRNA Isolation Kit (Ambion, Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. The RNA quality and quantity were determined using NanoDropTM1000 Spectrophotometer (Nanodrop ND-1000, Thermo Fisher Scientific, Wilmington, DE, USA). The integrity of total RNA (RIN value) was measured using the Agilent 2100 Bioanalyzer in combination with an Agilent 6000 Nano Chip (Agilent Technologies, Santa Clara, CA, USA). Extracted total RNA was stored at −80 °C.

Total RNA was initially extracted from 140 samples (70 HK and 70 LS samples) using the mirVana miRNA isolation kit (Ambion, Life Technologies, Carlsbad, CA, USA), in accordance with the manufacturer’s protocol. Extraction yield and RNA quality was measured on a NanoDrop1000 spectrophotometer (Nanodrop ND-1000, Thermo Fisher Scientific, Wilmington, DE, USA). RNA integrity (RIN value) was determined using an Agilent 2100 Bioanalyzer in combination with an Agilent 6000 Nano Chip (Agilent Technologies, Santa Clara, CA, USA). Following extraction, the total RNA was stored at −80 °C. Verification of infection was performed by PatoGen AS, Ålesund, Norway, according to their protocols. PatoGen is commonly used by Norwegian health authorities to measure M. viscosa load by qPCR.
A number of samples did not meet our quality thresholds, and together with the Ct-scores from the qPCR measurements this formed the basis for the final selection of samples to include in miRNA and mRNA expression studies. A total of 46 samples were selected; 22 HK, and 24 LS, including controls, all with RIN values larger than 9.0 (Table 1 and Table 2). Going forward, the LS samples were grouped by CT score rather than time point, to maintain homogeneity within each of the groups being compared (see Results).

Total RNA was isolated from human placenta using Trizol (Invitrogen, USA) according to the manufacturer’s directions. RNA quality was determined using Agilent 6000 Nano chips on an Agilent Technologies 2100 Bioanalyzer. Samples with RIN > 6 were selected for library construction. Sequencing libraries were constructed in a single batch according to the TruSeq SmallRNA Libary Preparation guide (Illumina) with no modification. In each sequencing lane on an Illumina HiSeq X10 platform, we pooled 14 samples with different index sequences and carried out 150-bp paired-ended sequences. We randomly distributed 10 African American, 10 European American, 10 South Asian American, and nine East Asian American samples into three sequencing lanes. We further included the tenth East Asian American sample (X14) into each of the three lanes to control for potential sequencing artifacts among the lanes (Additional file 1: Table S1).