Tapestation 4150

To assay the extent of angelicin modification, we selected the restriction endonuclease BciVI as its recognition sequence harbors a central ‘AT’ motif that becomes unrecognizable in the presence of an angelicin modification. Plasmid vector pBluescript SK+ (https://www.addgene.org/vector-database/1951/) was modified with 0, 100, and 500 uM angelicin prior to digestion with BciVI (NEB). 2.5 micrograms of DNA modified at each angelicin concentration were cut with either 4 or 8 units of restriction enzyme. Digests were analyzed on a 4150 TapeStation (Agilent) using a D5000 Screentape.

For scRNA-seq experiments, 5000 cells/replicate (n = 3 for day 7 and 21, n = 2 for day 14) at each time point (day 7, 14, and 21) were concentrated into 5 µL of cell resuspension buffer for PIP-seq (particle-templated instant partition sequencing; Fluent Biosciences), cells were captured, lysed and processed for library preparation as per the manufacturer’s instructions (PIPseq™ T2 3’ Single Cell RNA Kit v4.0). Briefly, mRNA was captured by breaking the stable emulsion via the addition of breaking buffer and cDNA was amplified and isolated from the PIPs by SPRI purification. Quality assessment and quantification of cDNA was achieved using a 4150 TapeStation (Agilent Technologies). cDNAs with values greater than 10 ng and peaks between the acceptable range for the high-sensitivity D5000 ScreenTape (100–5000 bp) were used for library preparation (fragmentation, end repair, A-tailing and adapter ligation) and sample index PCR was performed with unique dual index combinations. The final sequence-ready libraries were assayed for quality and quantity (size analysis of 35–1000 bp fragments via high-sensitivity D1000 ScreenTape) by Tape Station. Sequencing was performed on a NovaSeq 6000 (100-bp paired-end reads, Illumina) at the UCSD Institute for Genomic Medicine (IGM) Core to obtain >200 million reads per sample.

Following the manufacturer’s protocol, we isolated total RNA from dissected spinal cord samples using an RNeasy Mini Kit (Qiagen, Hilden, Germany) and quantified it with an ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). RNA quality was assessed with a 4150 TapeStation (Agilent Technologies, Santa Clara, CA, USA). Total RNA (50 ng) was then amplified, labeled with an Agilent Low-Input QuickAmp Labeling Kit (Agilent Technologies), and hybridized to SurePrint G3 Mouse GE microarray 8 × 60K ver. 2.0 (Agilent Technologies) according to the manufacturer’s protocols. All hybridized microarrays were scanned with a Microarray Scanner (Agilent Technologies). We calculated relative hybridization intensities and background hybridization values using Feature Extraction software (9.5.1.1) (Agilent Technologies). The gene array results were uploaded to the Gene Expression Omnibus repository (Accession number: GSE213844) at the National Center for Biotechnology Information homepage (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE213844 (accessed on 24 September 2022)).

Single-cell cDNA libraries were prepared using Single Cell 3′ Reagent Kit v3.1 and a 10× Genomics Chromium Controller. The number of cells in each channel of the Single-Cell Chip G did not exceed 5000. The dsDNA High Sensitivity kit measured the concentration of cDNA libraries on a Qubit 4.0 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). The quality of cDNA libraries was assessed using High Sensitivity D1000 ScreenTape on a 4150 TapeStation (Agilent, Santa Clara, CA, USA). The ready cDNA libraries were pooled, denatured, and sequenced on NextSeq 2000 (Illumina, San Diego, CA, USA) using pair-end reads (28 cycles for reading 1, 91 cycles for reading 2, and 8 cycles for the i7 index). Cell Ranger software (version 6.1.1) provided by 10× Genomics (version 6.1.1) (Pleasanton, CA, USA) was used to perform sample demultiplexing, alignment to the GRCh38 transcriptome, filtering, barcode counting, and UMI counting. The resulting datasets, M19 and ES36, were subjected to quality control. The subset was produced at a mark of 25% for mitochondrial material and recorded duplicates for each of the samples. Functional annotation was performed using R gProfiler2 gene ontology analysis [20 (link)].

100ng of total nucleic acid was used to prepare sequencing libraries with the Illumina RNA Prep with Enrichment kit and Illumina Respiratory Virus Oligo Panel. Library profiles and concentrations were evaluated on the Agilent 4150 TapeStation with the DNA1000 ScreenTape assay. Libraries with a size distribution of approximately 200bp to 700bp, with an average size of 350bp to 400bp, were pooled in equal mass and sequenced on an Illumina NextSeq 550 using PE74 reads with 5% PhiX spike-in. FASTQ files are available via NCBI accession PRJNA751858: SARS-CoV-2 Sequencing at Nemours Children’s Hospital Delaware.

Total RNA was extracted from each I. sinensis sample using the RNeasy Plus Mini Kit, and rRNA removal was performed using a RiBO-Zero Kit. Isolated RNA was used for cDNA library construction, using the dUTP method [74 (link)]. These libraries were sequenced on an Illumina HiSeq X Ten platform. The purity, concentration, and integrity of RNA were checked using the agarose gel electrophoresis, the Qubit 2.0 Fluorometer, and the Agilent 4150 TapeStation, respectively. After trimming adapters and filtering out low-quality reads, a total of 14.02 Gb clean reads were generated. The transcriptome was mapped to the reference genome using TopHat2 [75 (link)]. Transcripts greater than 200 bp in length and containing at least 2 exons were considered lncRNA candidates. Four computational approaches, including CPC [76 (link)], CNCI [77 (link)], Pfam (RRID:SCR_004726), and PhyloCSF [78 (link)], were combined to evaluate the protein-coding capability of the lncRNA candidates.

Total RNA was isolated using Direct-zol RNA MiniPrep Kit (Zymo Research, R2053) following manufacturer’s protocol. The concentration of total RNA was measured using Nanodrop one (Thermo Scientific, 701–058112) and 1 μg of total RNA was used for RNA-seq libraries constructed using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB England BioLabs, E7775) following manufacturer’s instruction. Briefly, rRNA was depleted from total RNA through probe hybridization method, and then fragmented and transcribed into cDNA. Through end repair/dA-tailing, adaptor ligation and PCR, the final libraries were quality controlled using Qubit 4.0 (Thermo Scientific, Q33238) and 4150 TapeStation (Agilent Technologies, G2992AA) for library concentration and size distribution, respectively. The libraries were sequenced using the Illumina HiSeq X Ten system using 2 × 150 bp paired-end sequencing strategy with 8 G bytes per cell.