For the positive-test group, we included all available RNA samples within 3 weeks of first positive SARS-CoV-2 PCR test, and convalescent samples at week 24 of follow-up for a subset of participants with available samples. For the control group, we included baseline samples only. Genome-wide mRNA sequencing was done as previously described30 (link) using the Kappa Hyperprep kit (Roche; Burgess Hill, UK) to generate complementary DNA libraries sequenced on the Illumina Nextseq platform using the Nextseq 500/550 High Output 75 cycle kit (Illumina; Cambridge, UK) according to manufacturers' instructions, giving a median of 26 million (range 19·8–32·4) 41-base pair paired-end reads per sample. We mapped RNAseq data to the reference transcriptome (Ensembl Human GRCh38 release 100) using Kallisto (version 0.46.1).31 (link) The transcript-level output counts and transcripts per million values were summed on gene level and annotated with Ensembl gene ID, gene name, and gene biotype using the tximport (version 1.20.0) and biomaRt (version 2.48.0) Bioconductor packages in R.32 , 33 (link)
Nextseq 500 550 high output 75 cycle kit
Manufactured by Illumina
Sourced in United Kingdom
The NextSeq 500/550 High Output 75 cycle kit is a laboratory equipment product used for DNA sequencing. It provides the necessary reagents and consumables for running high-throughput sequencing runs on the NextSeq 500/550 sequencing systems.
Automatically generated - may contain errors
Lab products found in correlation
Kappa hyperprep kit, by Roche (3 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (2 mentions)
Nextseq platform, by Illumina (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Nextseq 500, by Illumina (1 mentions)
Tempus spin rna isolation kit, by Thermo Fisher Scientific (1 mentions)
Globinclear kit, by Thermo Fisher Scientific (1 mentions)
Nextseq, by Illumina (1 mentions)
5 protocols using nextseq 500 550 high output 75 cycle kit
1
Longitudinal Transcriptomic Analysis of SARS-CoV-2 Infection
2
RNA Extraction and Sequencing Protocol
3
Transcriptomic Analysis of Skin Biopsies
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Three separate 3 mm punch biopsies were collected from each volunteer: one from normal (un-injected) skin, one from the saline injection site at 6 hours post-injection and one from the VZV injection site at 48 or 72 hours post-injection. Biopsies were immediately stabilised in RNAlater for cryostorage. Total RNA was extracted from bulk tissue homogenates using RNeasy Mini Kit (Qiagen) as previously described [8 (link)]. Library preparation for RNAseq was performed using the Kappa Hyperprep kit (Roche Diagnostics) and sequencing was performed by the Pathogens Genomic Unit (UCL) on the Illumina Nextseq 500 (Illumina) using the Nextseq 500/550 High Output 75 cycle kit (Illumina) according to the manufacturers’ instructions, resulting in a median of 22.7 million (range 1.4–38.6 million; IQR 20.8–24.4 million) 41 bp paired-end reads per sample.
4
RNA Extraction and Sequencing Protocol
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Total RNA was extracted with the Qiagen AllPrep DNA/RNA Mini Kit (Hilden, Germany) and analyzed with Nanodrop spectrophotometer for concentration and purity. RNAseq was performed at the University of Pittsburgh Genomic Core using NextSeq 500/550 High Output 75 cycle kit (Illumina, San Diego, CA) with a single-read length and 40 million read depth.
5
Transcriptional Profiling of COVID-19 Blood RNA
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Blood samples for RNA sequencing were collected in Tempus Blood RNA tubes. For ‘cases’, we included all available RNA samples, including convalescent samples at week 24 of follow-up for a subset of participants. For uninfected controls, we included baseline samples only. Genome wide mRNA sequencing was performed as previously described.28 (link) Total blood RNA was extracted using the Tempus Spin RNA Isolation Kit (ThermoFisher). Globin mRNA and genomic DNA were removed using the GlobinClear kit (ThermoFisher) and the TURBO DNA-free kit (ThermoFisher) respectively. Transcriptional profiling of blood RNA samples was performed by RNA sequencing. cDNA libraries were generated using the Kappa Hyperprep kit (Roche), and sequencing was performed on the Illumina Nextseq using the Nextseq 500/550 High Output 75 cycle kit (Illumina) according to manufacturers' instructions, resulting in a median of 26 million (range, 19·8–32 · 4 million) 41 bp paired-end reads per sample. RNAseq data were mapped to the reference transcriptome (Ensembl Human GRCh38 release 100) using Kallisto.29 (link) The transcript-level output counts and transcripts per million (TPM) values were summed on gene level and annotated with Ensembl gene ID, gene name, and gene biotype using the R/Bioconductor packages tximport and BioMart.30 ,31 (link)
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