Caliper labchip xt

DNA (500ng) was fragmented using a Covaris S2 Focused ultrasonicator to a mean size of 300 bp. Fragment ends were repaired and phosphorylated with T4 DNA polymerase (New England Biolabs, USA), Klenow fragment (New England Biolabs, USA), and T4 polynucleotide kinase (New England Biolabs, USA). A 3’ A-base overhang was introduced with Klenow exo-minus (New England Biolabs, USA) and fragments were ligated to Illumina paired-end adaptors. Ligation products with a mean size of 350 bp +/- 20% were isolated on a Caliper LabChip XT (PerkinElmer) and amplification was performed using Illumina PCR primers InPE1.0 and InPE2.0 and primer indices. Pooled, indexed libraries were captured using an Agilent SureSelect Custom DNA kit targeting exons of 197 commonly mutated cancer genes (Agilent Technologies, USA) according to the manufacturer’s protocol and sequenced at a depth of 67x and 163x for SB.07 and SB.06, respectively. Alignments to the hg19 human reference genome assembly were performed with BWA version 0.6.2-r126 [22 (link)] and duplicates were marked with picard [23 (link)]. Single nucleotide variants were called with the samtools package [23 (link)] and small insertions and deletions were called with pindel version 0.2.5 [24 (link)].

Initial small-RNA libraries were prepared from 1 μg total RNA (TruSeq Small RNA kit, Illumina), followed by miRNA enrichment (Caliper LabChipXT, PerkinElmer) according to manufacturer’s protocols. Small RNA-Seq libraries were sequenced on Illumina HiSeq 2000. Library preparation and sequencing were conducted in FIMM Sequencing Laboratory, University of Helsinki, Finland. Quality control of the raw reads was performed using FastQC (ver. 0.11.7) and MultiQC (ver. 1.7) (Ewels et al., 2016 (link)). Trimmomatic (ver. 0.38) was implemented to remove adapters and trim the quality of reads with the following settings - ILLUMINACLIP:2:30:9, LEADING:3, CROP:50, TRAILING:3, SLIDINGWINDOW:4:20, MINLEN:16. Reads were aligned to human genome reference GRCh38 using bowtie (ver. 1.2.2, settings: -n 1 -l 20 -q -m 40 -k 1 -t –best –strata) (Langmead et al., 2009 (link)). miRNA quantification was performed using featureCounts from the Rsubread package (ver. 1.20.6) (Liao et al., 2019 (link)) for R with miRNA annotations from miRBase 22.1 as reference (Kozomara et al., 2019 (link)).

To measure 5-hydroxymethylcytosine (5hmC) in MO and moDC across the genome, we used the Hydroxymethyl Collector kit (Active Motif) according to the manufacturer’s instructions. Captured DNA fragments were subjected to library preparation as follows. DNA was end-repaired, A-tailed, adapter ligated (NEXTflex DNA Barcodes), and purified using magnetic beads (Agencourt AMPure XP). Libraries were PCR-amplified (4 cycles), bead-purified, and size-selected on the Caliper LabChip XT (PerkinElmer) using the LabChip XT DNA 300 assay kit (PerkinElmer) according to the manufacturer’s instructions. After size selection, DNA was subjected to another 12 cycles of PCR amplification and purified using magnetic beads (Agencourt AMPure XP). The quality of dsDNA libraries was analyzed using the High Sensitivity D1000 ScreenTape Kit (Agilent) and libraries were sequenced on a HiSeq1000 sequencer (Illumina). Sequencing libraries are listed in Supplementary Table 4.

The four johnsongrass samples were run through a denaturing agarose gel to visualize RNA integrity (Supporting Information Figure S1). Samples 3 and 4, which depicted intact RNA, were pooled and submitted for library prep with the TruSeq Stranded mRNA Library Prep kit (Illumina). The resulting cDNA library was sequenced in a single lane of High Output (2 × 125 bp) on the Illumina HiSeq 2500 platform. The raw reads were trimmed in BBduk (Bushnell, 2014), removing adaptors and reads that were short or low quality.
Thinopyrum intermedium rhizome RNA libraries were prepared using the Illumina TruSeq RNA Library Prep Kit v2 by the University of Minnesota Genomics Center and sequenced across three lanes of an Illumina HiSeq 2000 (2 × 100 bp) run. Libraries were size‐selected with a Caliper LabChip XT (PerkinElmer) to have an approximate insert size of 200 bp. The resulting reads were also trimmed with bbduk to produce 5.7 gigabases of high‐quality (>Q30) data as 65,413,146 paired‐end reads (NCBI Sequence Read Archive SRX3529031).