Hiseq 2500 rapid run platform

Small RNA sequencing was performed on pools of 5 individual RNA samples from each group sampled at 0, 12, 36 and 60 hpi, with the exceptions; PBS control group only sampled at 0 and 36 hpi and reduced sample in VP28 specific dsRNA treatment group at 60 hpi (n = 4). Each individual RNA sample contributed with an equal amount of total RNA mass per pool based on Picodrop measurements, and each pool was aimed to end up with a final RNA concentration of 200 ng/µl, although a repeated measurement performed on prepared pools did show the average RNA concentration to be 195 ng/µl ± 11 SD (ng/µl range [173, 208]). Small RNA sequencing was performed at Theragenetex (Seoul, Korea), and consisted of RNA sample quality control (Bioanalyzer), library preparation with a TruSeq® Small RNA Library Prep Kit (Illumina) and 50 SE sequencing with 20 M reads per sample on a HiSeq2500 rapid run platform (Illumina). The sequencing was performed according to Illumina’s standard ISO protocol.

The proband's DNA was collected, sequenced, and analyzed as part of a larger study developing a targeted multi‐disorder high‐throughput sequencing assay. The methods have been detailed previously (Delio et al. 2015). In brief, ~10 mL of whole blood was collected from the patient and genomic DNA was purified using the Puregene Genomic DNA Purification kit (Gentra, Minneapolis, MN, USA). Next, all coding, untranslated regions (UTR) and flanking intronic regions of 650 known disease‐associated genes were targeted. Within this panel there were 154 cardiac disease‐associated genes (listed in Table S1). Targeted capture‐sequencing was done using the Roche‐NimbleGen EZ SeqCapV3 capture system and sequenced on the Illumina Hiseq 2500 Rapid Run platform. Sequence reads were analyzed using a custom in‐house generated analytical pipeline (Delio et al. 2015). We sequenced eight controls and one HapMap sample to assess the precision of the panel in identifying known variants. Samples were analyzed for copy number variation using the Affymetrix Genome Wide SNP Array 6.0. Sanger sequencing was used to confirm mutations. All mutations discovered were subsequently validated in a CLIA‐approved commercial laboratory.

RNA was isolated using Qiagen RNA–DNA allprep columns or TriReagent (Sigma) and treated with DNase I (Ambion DNA-free DNA [1311027] or Thermo Fisher RNase-free [EN0525]) following the manufacturer's instructions. cDNA was generated using 0.5–1 µg of RNA (Thermo RevertAid, K1622), and qRT–PCR was performed using Brilliant III SYBR master mix (Agilent Technologies, 600882). Relative quantification was performed using the comparative CT method with normalization to CycloB1 levels. Primer sequences are available on request. Opposite strand-specific polyA RNA libraries were made using 1 µg of DNase-treated RNA at the Sanger Institute Illumina bespoke pipeline and sequenced as single-end 50-base-pair (bp) reads using the Illumina HiSeq 2500 Rapid Run platform.

RNA was isolated using RNeasy Mini kit (Qiagen, 74104) and treated with DNaseI (Thermo Fisher Scientific, EN0521) following manufacturer’s instructions.
For SAM and E14 ESCs, opposite strand-specific total RNA libraries (ribozero) were made from 1 μg of DNase-treated RNA using the Sanger Institute Illumina bespoke pipeline and sequenced at 100 bp paired-end on the Illumina HiSeq2500 Rapid Run platform. For CRISPRa (see Table S1 for sgRNAs used) and cDNA validation samples, opposite strand-specific polyA-capture RNA libraries were made from 1 μg of DNase-treated RNA using the Sanger Institute Illumina bespoke pipeline and sequenced at 50 bp single-end on Illumina HiSeq4000.
All bulk RNA-sequencing experiments were performed in three independent replicates, except for SAM ESCs for which two replicates were prepared.

ATAC-seq was performed as previously described [72 (link)]. Briefly, two e9.5 placentas were microdissected from timed-pregnant CD-1 mice (Charles Rivers Labs) for each biological replicate (3 replicates in total). After tissue homogenization and cell lysis, we followed the transposition and purification steps as described previously [72 (link)]. We amplified the purified DNA for 12 cycles following conditions specified in [72 (link)] using adapters from the Nextera index kit (Illumina) (Additional file 2: Table S3). PCR purification was performed using AMpure XP beads (Beckman Coulter) in order to remove large fragments and remaining primers. Library quality was assessed using the Bioanalyzer High Sensitivity DNA Analysis kit (Agilent) to check for proper periodicity, and the DNA concentrations were estimated using the Qubit and Bioanalyzer (Additional file 1: Figure S1a). Libraries were sequenced by Elim Biopharmaceuticals, Inc. using the Illumina HiSeq2500 Rapid run platform with 50 bp paired-end sequencing. Before filtering, there was an average of about 82 million reads (73–93 million), and after filtering there was an average of about 18.8 million reads (16–20 million) (Additional file 2: Table S4).

A chemical genomic analysis of grape juice medium was performed as described in Piotrowski et al. (2015) . Briefly, 200 µl cultures of a pooled collection of S. cerevisiae deletion mutants were grown in grape juice supplemented with amino acids (which improves lab-strain growth in grape juice [Harsch et al. 2010 (link)]), or a YPD control, for 48 h at 30 °C. DNA was extracted using the Epicentre MasterPure Yeast DNA purification kit. Mutant-specific barcodes were amplified with multiplex primers containing Illumina adapters (Smith et al. 2009 (link)). The barcodes of four replicates of each condition (grape juice vs. YPD) were sequenced using an Illumina HiSeq2500 Rapid Run platform. Differential abundance and significance were assessed for barcodes with at least ten reads using edgeR (Robinson et al. 2010 (link)). Functional enrichment was performed using FunSpec (Robinson et al. 2002 (link)). Fourteen of the 740 significant genes (FDR 0.05) fell under the 90% credible interval of grape juice QTL. Enrichment for genes under QTL peaks was assessed by randomly sampling 740 genes from the yeast genome and counting the frequency from 10,000 trials in which 14 or more genes fell under QTL peaks (supplementary table S5, Supplementary Material online).