Nextseq platform

Total RNA was extracted and treated with BioRobot^® EZ1 and RNA Tissue Mini Kit (Qiagen, Hilden, Germany) as previously described by Le, Shao (48). RNA quantity and integrity were validated using a NanoDrop ND-1000 UV—vis Spectrophotometer (NanoDrop Technologies, Wilmington, USA) and Agilent 2100 Bioanalyzer and RNA 6000 Nano LabChip kit (Agilent Technologies, Palo Alto, USA) respectively. All samples had 260/230 and 260/280 ratios above 2.0 and 2.2 respectively. The average RNA integrity number (RIN) of all samples was 7.9±0.7. Sequencing and library preparation were performed by the Norwegian Sequencing Centre (www.sequencing.uio.no). DNA libraries were prepared as previously described by [46 (link)] using 90 ng total RNA input to the TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, California, USA). For multiplexing, standard Illumina adaptors were used. The libraries were sequenced using the NextSeq Illumina platform (Illumina, San Diego, California, USA) according to the manufacturer’s instructions, generating single end 75bp read libraries with an average library size of 25±6 million reads. Raw reads were submitted to the gene expression omnibus https://www.ncbi.nlm.nih.gov/geo/ (accession number GSE129459).

Frankia strains AgB32 and AgKG'84/4 were grown from stocks preserved in 20% vol/vol glycerol at -80 °C since 2003 in Defined Propionate Medium (DPM) containing propionate and NH₄Cl as C and N source, respectively (19), at 30 °C for two weeks. Cells were harvested by centrifugation (15,000 × g, 5 min) and homogenized through sonication (10 s at 20% output in a S-450 sonifier, Branson Ultrasonics, Danbury, CT) 20 (link). DNA was extracted from cell pellets after an additional centrifugation step using the SurePrep^TM Soil DNA Isolation Kit (Fisher Scientific, Houston, TX) 21 (link), and concentrations measured with a Qubit^® 2.0 Fluorometer (Life Technologies, Carlsbad, USA). Library preparation and sequencing using the Illumina tagmentation protocol and the NextSeq Illumina platform (2 × 150 bp) using standard protocols were done at the Microbial Genomics Sequencing Center, Pittsburgh, PA, USA.

Strand-specific libraries were generated as previously described^{10 (link)}, from 192 barcoded single-cells and sequenced on a NextSeq Illumina platform. The 192 barcoded single-cell libraries were pooled for sequencing of the HG002 sample. Raw demultiplexed fastq files from the paired-end sequencing run (80-bp read lengths) were uploaded for each single-cell library. These data can be found at https://s3-us-west-2.amazonaws.com/human-pangenomics/index.html?prefix=HG002/hpp_HG002_NA24385_son_v1/Strand_seq/.

A brain sample was fragmented and passed through an insulin needle. The homogenate was clarified by centrifugation and filtered with a 0.45 µm syringe filter. The filtrate was digested with Turbo DNase (Ambion) and RNase I (Invitrogen) to remove non-protected human nucleic acids. Remaining nucleic acids were extracted using the QIAamp viral RNA minikit (Qiagen). Purified RNA was reverse-transcribed with Superscript III reverse transcriptase (Invitrogen) and second-strand DNA synthesis was performed with Klenow fragment polymerase (New England Biolabs) using barcoded primers consisting of a 20-nucleotide–specific sequence upstream of a random nonamer, as previously described [18 (link)]. The resulting DNA products were PCR amplified, and libraries were constructed with the Nextera XT DNA library preparation kit (Illumina) and sequenced on a NextSeq Illumina platform (2 × 150 bp run).

For scRNAseq, InDrop (Klein et al., 2015 (link)) was implemented following the protocol as previously described (Zilionis et al., 2017 (link)). Briefly, stage 7 islet-like aggregates were dissociated into single cells by incubation with a 1:1 mixture of TrypLE Express and Trypsin-EDTA for 10 min at 37°C. Dissociated cells were passed through a 30 μm strainer to remove cell clumps. Single cells were co-encapsulated into 3–4 nL droplets together with barcoded hydrogel beads and a mixture of reverse-transcription (RT) and lysis reagents. Within every single droplet, a cell was lysed and cDNA tagged with a barcode during reverse transcription. The droplet emulsion was broken and the bulk material was taken through the following steps: i) second strand synthesis; ii) linear amplification by in vitro transcription (IVT); amplified RNA fragmentation; iv) reverse transcription; v) PCR. In total, four samples from independent experiments were processed. They were produced in two parallel differentiation runs from HEL71.4 mutant and HEL71.4-A2 corrected iPSC lines, that were encapsulated at two different timepoints. The resulting DNA libraries were multiplexed and sequenced together on NextSeq Illumina platform in paired-end mode using a high-yield 75 cycle kit. Read quality was assessed by running FASTQC (version 0.10.1).

RNA-Seq libraries were constructed as in Tsuda et al.^{59 (link)} except that 6 µg of total RNA was used rather than 3 µg. Three libraries were made of four pooled 9–11 mm tassels: two mutants and one wild type. Libraries were sequenced on a NextSeq Illumina platform with 75 bp paired-end reads. Raw reads were aligned to the maize B73 genome AGPv3.30 using HISAT2^{60 (link)} and counted to AGPv3.30 gene models using HTSeq-counts^{61 (link)} using the cyberinfrastructure provided by Cyverse Atmosphere^{62 (link)}. Reads were visualized using the Integrative Genomics Viewer (Broad Institute)^{63 (link)}. Counted reads were tested for differential expression with edgeR using a generalized linear model (GLM) approach on transcripts with a raw count greater than 5 in at least one condition and FDR significance threshold of 0.05^{64 (link),65 (link)}. Differentially expressed genes (FDR ≤ 0.05) between Ts5 and WT siblings were separated by -log(fold-change) into upregulated and downregulated differentially expressed gene lists. Gene accessions from each list were tested for Gene Ontology term enrichment by singular Gene Ontology term enrichment analysis (SEA) with agriGO v2.0^{66 (link)}. Quantitative reverse transcription PCR was performed as in Bolduc et al.^{67 (link)}.