RNA was isolated from sorted cells using either the NucleoSpin RNA Kit (Macherey-Nagel) or TRIzol reagent (Thermo Fisher Scientific). RNA-seq libraries were prepared from two biological replicates using the True-Seq Stranded Total RNA Kit (Illumina) and sequenced paired-end in a pool of 12 indexed libraries using a Next-Seq 500/550 High Output Kit v2 150 cycles (Illumina) at the Genomics Birmingham sequencing facility.
Nextseq 500 550 high output kit v2 150 cycles
Manufactured by Illumina
The NextSeq 500/550 High Output Kit v2 (150 cycles) is a laboratory equipment product designed for use with Illumina's NextSeq 500 and NextSeq 550 sequencing systems. The kit provides the necessary reagents and consumables to perform 150 cycles of high-output sequencing on the respective platforms.
Automatically generated - may contain errors
Lab products found in correlation
Truseq stranded mrna library prep kit high throughput, by Illumina (3 mentions)
Qubit dna hs assay kit, by Thermo Fisher Scientific (3 mentions)
Dna 100 kit, by Agilent Technologies (3 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (3 mentions)
Nextseq 550, by Illumina (3 mentions)
Agencourt ampure xp bead, by Beckman Coulter (3 mentions)
Chromium controller, by 10x Genomics (2 mentions)
Nextseq 500 instrument, by Illumina (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions)
Trueseq stranded total rna kit, by Illumina (1 mentions)
Nucleospin rna kit, by Macherey-Nagel (1 mentions)
Truseq stranded mrna ht kit, by Illumina (1 mentions)
Nextseq 500 system, by Illumina (1 mentions)
Agilent 4200 tapestation, by Agilent Technologies (1 mentions)
D1000 screentape, by Agilent Technologies (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Rna screentape, by Agilent Technologies (1 mentions)
Chromium single cell 3 reagent kit v2, by 10x Genomics (1 mentions)
9 protocols using nextseq 500 550 high output kit v2 150 cycles
1
RNA-seq Library Preparation and Sequencing
2
RNA Sequencing Library Preparation
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Total RNA was extracted using an RNeasy mini kit (Qiagen) according to the manufacturer’s protocol. The RNA concentration was measured using NanoDrop (Thermo Fisher Scientific), and the quality of RNA was analyzed using the Agilent 4200 TapeStation (Agilent, Santa Clara, CA, USA), RNA Screen Tape (Agilent), and RNA reagent kit (Agilent), after which the samples were stored at −80 °C. The method of the confirmation of RNA quality was in accordance with the manufacturer’s instructions. The RNA was used to prepare a cDNA library for RNA sequencing. TruSeq Stranded mRNA HT kit (Illumina, San Diego, CA, USA) was used for library preparation. The concentration of the prepared library was measured by Qubit (Thermo Fisher Scientific). The Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) was used, and the method was performed according to the kit manual. The length of the DNA fragment of the library was analyzed by the Agilent 4200 TapeStation. The DNA fragment length was determined using the D1000 ScreenTape (Agilent) and the D1000 reagent kit (Agilent). Each library was stored at −20 °C. The libraries with confirmed quality were sequenced with pair-end sequencing using the NextSeq 500 System (Illumina) and the NextSeq 500/550 High Output Kit v2 (150 Cycles) (Illumina). The library preprocessing was performed according to the manufacturer’s instructions.
3
High-Throughput RNA-Seq Library Preparation
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Libraries were prepared using the TruSeq Stranded mRNA Library Prep Kit, High Throughput (Illumina, San Diego, CA) with Superscript III Reverse Transcriptase (Life Technologies, Carlsbad, CA) and Agencourt AMPure XP beads (Beckman Coulter, Brea, CA). Libraries prepared were quantified using the Qubit DNA HS Assay kit (Thermo Fisher) and quality was assessed using the DNA 100 kit (Agilent Technologies), run on a 2100 Bioanalyzer. 75bp, paired-end RNA-sequencing was performed on the NextSeq 550 using the NextSeq 500/550 High Output Kit v2 (150 cycles) (Illumina). Each flow cell contained 12 libraries.
4
Single-cell RNA-seq of LCMV-specific CD8 T Cells
5
High-Throughput RNA-Seq Library Preparation
6
Preparation of DNA and RNA Libraries
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DNA libraries from DNase I digestions and chromatin immunoprecipitation assays were prepared using the Kapa Hyper‐Prep kit according to the manufacturer's guidelines. Libraries were indexed and then amplified by PCR. For DNase‐Seq, libraries were amplified for 12 PCR cycles, and for ChIP‐Seq, libraries were amplified for 16 cycles. DNA fragments from amplified libraries were agarose gel electrophoresis purified to ensure the correct fragment lengths were sequenced (Qiagen Gel purification Kit). Fragments of 200–300 bp were selected for DNase‐Seq libraries and 200–400 bp for ChIP libraries. Libraries were validated by qPCR and quantified using the Kapa quantification kit before pooling and sequencing on NextSeq® 500/550 High Output kit v2 75 cycles (Illumina, FC 404‐2005).
RNA libraries were prepared from 50 to 100 ng RNA using the NEB Next® Ultra™ II Directional RNA library Prep kit for Illumina (NEB, #E7760S) according to the manufacturer's instructions. rRNA was depleted using the NEBNext® rRNA Depletion kit (NEB, #E6310S). Libraries were quantified using the Kapa quantification kit before pooling and sequencing on NextSeq® 500/550 High Output kit v2 150 cycles (Illumina, FC 404‐2002).
RNA libraries were prepared from 50 to 100 ng RNA using the NEB Next® Ultra™ II Directional RNA library Prep kit for Illumina (NEB, #E7760S) according to the manufacturer's instructions. rRNA was depleted using the NEBNext® rRNA Depletion kit (NEB, #E6310S). Libraries were quantified using the Kapa quantification kit before pooling and sequencing on NextSeq® 500/550 High Output kit v2 150 cycles (Illumina, FC 404‐2002).
7
Single-cell RNA-seq analysis of CD4 T cells
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CD4 T cells were isolated from single-cell suspensions of splenocytes using a negative selection isolation kit (StemCell), then FACS-sorted (see above) for CD44+CD4 T cells. 10,000 cells per sample were loaded onto the Chromium Controller (10x Genomics), then the 10x Genomics Chromium Next GEM Single Cell 5′ Reagent Kits v2 (Dual Index) was used to generate cDNA and HTO libraries, as described in the manufacturer’s protocols. Barcoded libraries were sequenced on an Illumina NextSeq 500 instrument using a NextSeq 500/550 High Output Kit v2 (150 cycles) (FC-404–2002, Illumina) with the following cycle counts: 28 (read 1), 10 (index 1), 10 (index 2), 90 (read 2). Data were demultiplexed and aligned to the mm10 2020-A reference transcriptome (10x Genomics) using Cell Ranger (v6.0, 10x Genomics). Analysis was performed in R using Seurat (v4.0.6).62 (link),63 (link) Number of genes detected per cell and percent mitochondrial genes were plotted, and outliers were removed (number of genes over 2,500 and under 200, and percent mitochondrial genes over 10%) to filter out doublets and dead cells. Samples were integrated and PCA and UMAP were performed using the top 30 principal components.
8
Spatial Transcriptomics of Mouse Spleen Tissue
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10 μm sections were generated from frozen spleens using a cryostat and placed on barcoded slides (10x Genomics). The Visium Spatial Gene Expression Slide & Reagent Kit (10x Genomics) was used to generate barcoded cDNA libraries, as described the manufacturer’s protocols. H&E stained tissues on Visium slides were imaged using a Nikon Eclipse Ti2 inverted microscope. Spleen tissue on Visium slides was permeabilized for 18 min to extract mRNA. Barcoded cDNA libraries were sequenced on an Illumina NextSeq 500 instrument using a NextSeq 500/550 High Output Kit v2 (150 cycles) (FC-404–2002, Illumina) with the following cycle counts: 28 (read 1), 10 (index 1), 10 (index 2), 90 (read 2). Loupe Browser (v5.0, 10x Genomics) was used to identify which spatial sequencing capture area spots were in contact with tissue. Demultiplexing and alignment was performed with Space Ranger (v2.1, 10x Genomics) and the mm10 2020-A reference transcriptome (10x Genomics). Analysis was performed in R using Seurat (v4.0.6). Visium datasets were integrated using the SCTransform pipeline, and PCA and UMAP were performed using the top 30 principal components. SPOTlight43 (link) was used to demultiplex Visium data with our integrated reference scRNA-seq data used as a reference.
9
RNA-seq Library Preparation and Sequencing
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Libraries were prepared using the TruSeq Stranded mRNA Library Prep Kit, High Throughput (Illumina, San Diego, CA, USA) with Superscript III Reverse Transcriptase (Life Technologies, Carlsbad, CA, USA) and Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA). Libraries were quantified using the Qubit DNA HS Assay kit (Thermo Fisher), and quality was assessed using the DNA 100 kit (Agilent Technologies), run on a 2100 Bioanalyzer. 75‐bp, paired‐end RNA‐sequencing was performed on the NextSeq 550 using the NextSeq 500/550 High Output Kit v2 (150 cycles) (Illumina). Each flow cell contained 12 libraries.
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