Nextseq500v2 instrument

Total RNA was isolated following the procedure of Reid et al. (2006) (link) using a CTAB- spermidine extraction buffer. The RNA was quantified using NanoDrop 1000 spectrometer (Thermo Fisher Scientific). The integrity of RNA was assessed by an Agilent 2100 Bioanalyzer using a sRNA chip (Agilent Technologies) according to the manufacturer’s instructions.
sRNA libraries were prepared using total RNA as input (1 µg) according to the instructions provided by TruSeq sRNA Sample Preparation Kit (Illumina). Twenty-eight bar-coded sRNA libraries were constructed. The quality of each library was assessed using an Agilent High Sensitivity DNA chip on an Agilent 2100 Bioanalyzer. Equi-molar concentrations of libraries were pooled and sequenced on an Illumina NextSeq500v2 instrument at the Institute for Integrative Genome Biology, UC Riverside. One library (minus UV-B, -3 WAV, high-fluence UV greenhouse experiment) was re-sequenced to increase reads depth on a MiSeqv3 platform, and another library was solely sequenced on MiSeqv3 (plus UV-B, +6 WAV, field grown; Supplementary Table 1a). This latter library had consequently fewer reads and the 21 and 24 nt species of small RNAs were not as abundant due to overrepresentation of a specific 48 nt non-coding RNA homologous mapping to chromosome 11 intergenic region.

The three Danish couples and one infant were exome sequenced at 100x coverage. Briefly, the library was constructed using an Agilent SureSelectXT Human AllExon 60 Mb enrichment kit and NuGEN library preparation. The sequencing was done on a NextSeq 500 v2 instrument (Illumina, CA, USA) with 2x150 bp reads. Alignment of quality trimmed reads against the reference genome hg19 was done using, Burrows–Wheeler alignment—MEM, maximal exact match.
BWA-MEM v 0.7.12. Variant discovery of samples was done with FreeBayes v1.0.2. IGV software was used for viewing BAM and VCF files. Second-level analysis was done by Ingenuity software (Ingenuity, Qiagen, Copenhagen) after uploading of VCF files. The analysis was done as trio-analysis and as two-group analysis and a combination of analysis of the individual couples searching for homozygotic variants, compound heterozygotic variants, and de novo mutations. The question was if the women predictably would not express an antigen that the fathers would pass on to the child allowing for maternal antibodies to target such a hypothetical alloantigen on fetal liver cells.