For droplet-based scRNA-seq, the tissues of the endometrium (N = 3) were pooled. Cells were counted, and ∼13,000 cells were loaded per channel onto a chromium controller (10× Genomics) for the generation of gel bead-in-emulsions. Sequencing libraries were prepared using Single Cell 3′ Reagent Kits v3 (10× Genomics) and then converted using the MGIEasy Universal Library Conversion Kit (BGI) before sequencing on a MGISEQ-2000 instrument (BGI). For the BGI FASTQ files to be made compatible with the “cellranger count” pipeline from Cell Ranger version 3.0.2 (10× Genomics), the file names and the FASTQ headers were reformatted using the code from https://github.com/IMB-Computational-Genomics-Lab/ BGIvsIllumina_scRNASeq. Data were processed using Homo_sapiens GCF_000001405.39_GRCh38.p13 as a reference.
Single cell 3 reagent kits v3
Manufactured by 10x Genomics
Sourced in United States
The Single Cell 3' Reagent Kits v3 are laboratory equipment used for single-cell RNA sequencing. They provide the necessary reagents and materials to prepare single-cell samples for transcriptome analysis.
Automatically generated - may contain errors
Lab products found in correlation
Novaseq 6000, by Illumina (3 mentions)
Chromium controller, by 10x Genomics (1 mentions)
Agilent 4200 tapestation system, by Agilent Technologies (1 mentions)
Nextseq 2000 platform, by Illumina (1 mentions)
Qubit dsdna high sensitivity assay kit, by Thermo Fisher Scientific (1 mentions)
Nextseq 500 system, by Illumina (1 mentions)
Nextseq 500, by Illumina (1 mentions)
Chromium single cell controller, by 10x Genomics (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
Mirneasy micro kit, by Qiagen (1 mentions)
Nextseq 2000, by Illumina (1 mentions)
9 protocols using single cell 3 reagent kits v3
1
Droplet-based scRNA-seq of Endometrial Tissue
2
Single-nucleus RNA-seq of Nrf1-deficient hearts
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Tail genotyping was performed on the day of collection (P4, one day post-P3 surgery) to distinguish Nrf1fl/fl and Nrf1fl/fl:αMHC-Cre mice. Mice were then euthanized, and hearts were extracted. Each heart was dissected in ice-cold PBS and a transverse cut on the ligation plane (for MI hearts) or similar level (for sham hearts) was made. Heart tissue below the ligation plane was collected and pooled from 4 to 6 hearts of the same genotype for nuclei isolation. Cardiac nuclei isolation was performed as previously described51 (link). Total cardiac nuclei were used to generate single nucleus RNA-seq libraries using Single Cell 3′ Reagent Kits v3 (10×Genomics) according to the manufacturer’s protocol.
3
Single-cell RNA-seq of thawed samples
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Single-cell barcoding of thawed samples and complementary DNA (cDNA) library preparation were performed according to the manufacturer's protocol (Single Cell 3′ Reagent Kits v3, 10x Genomics, USA). Quality control was maintained using a Bioanalyzer High sensitivity DNA chip (Agilent 4200 TapeStation System, Amstelveen, Netherlands) and Qubit dsDNA High Sensitivity Assay Kit (Thermo Fisher, Waltham Massachusetts, US) before library preparation. The cDNA libraries were sequenced on a NextSeq 2000 Illumina platform (Illumina, Inc., San Diego, CA). Base call files were demultiplexed into FASTQ files using Cell Ranger's (v7.1.0) mkfastq pipeline. The Read2 files were trimmed using cutadapt (v2.7), and reads shorter than 20 bp were removed. Read processing was conducted with zUMIs v2.0 pipeline, and trimmed reads were aligned to GRCh38 (GRCh38.p13) using STAR (v2.7.2a). We removed barcoded cells with <100 transcripts and cells with >20% of their transcriptome of mitochondrial origin (Figure S1 A).
4
Cardiac Single Nuclei Isolation and RNA-seq
5
Single-cell RNA Sequencing with 10X Genomics
6
Single-cell RNA-seq of Mouse Fibroblasts
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SIST cells were prepared in accordance with the protocol described in this study. Between 70,000 and 100,000 mouse adherence-cultured fibroblasts (monolayer) and dissociated spheroid cells (single cells) were freshly prepared and their gene expression profile analyzed using single-cell 3′ RNA-sequencing (StemCore Laboratories, Ottawa General Hospital, U Ottawa). Sequencing libraries were prepared using Single Cell 3′ Reagent Kits V3.1 (10x Genomics, Pleasanton, CA, USA) with the 10x Chromium controller, and sequenced on NextSeq 500 (Illumina, San Diego, CA, USA). Library construction, sequencing and initial analysis were performed by StemCore Laboratories (Ottawa Hospital Research Institute, University of Ottawa).
7
Single-cell RNA-seq of CXCR6-/- OT-1 T cells
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Ears of C57Bl/6J mice were infected with VACV-OVA the day after receiving 15,000 WT OT-1 T cells and 15,000 CXCR6−/− OT-1 T cells. At 21 days post infection, lymphocytes were isolated from skin after intravenous injection of anti-CD45-PE antibody as previously described. WT OT-1 T cells (CD8+CD90.2+CD44+CD45 IV−CD45.1+CD90.1−) and CXCR6−/− OT-1 T cells (CD8+CD90.2+CD44+CD45 IV−CD45.1−CD90.1+) were sorted. The sorted cellular suspensions were loaded on a 10x Genomics Chromium instrument to generate single-cell gel beads in emulsion. Libraries were prepared using Single cell 3’Reagent kits v3.1 (Chromium Next GEM Single Cell 3’ GEM, Library & Gel Bead Kit v3.1, 16 rxns PN-1000121;10x Genomics) and were sequenced using Illumina Novaseq 6000.
8
Single-Cell RNA Sequencing of Biomphalaria glabrata
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Single cell processing procedure is defined by the MGX platform (Montpellier GenomiX). Single cell suspension was obtained by a Chromium Single-Cell Controller (10X Genomics). Library preparation was performed with Single Cell 3’ Reagent kits V3.1 (10X Genomics) using 10x Next GEM Technology barcode and validated by DNA quantification with Fragment Analyzer (kit High Sensitivity NGS) and qPCR (ROCHE LightCycler 480). Libraries were sequenced with an illumina NovaSeq 6000 (Illumina) and SBS (Sequence By Synthesis) techniques using NovaSeq Reagent kits (100 cycles). Output results and matrix generation were processed with 10X Genomics Cell ranger v3.1.0 software (http://10xgenomics.com ).
All available mitochondrial gene sequences were recovered from the NCBI database (34 (link), 35 (link)) and gene names corresponding to these sequences (Supplementary Table 2 https://vectorbase.org/vectorbase/app/record/dataset/TMPTX_bglaBB02 , 15/10/2021) (37 (link)).
All available mitochondrial gene sequences were recovered from the NCBI database (34 (link), 35 (link)) and gene names corresponding to these sequences (
9
Bulk and Single-Cell RNA-Seq of G+ Cell Samples
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Total RNA was isolated from five paired samples (G+/G−) using the miRNeasy Micro kit (Qiagen, Germany), according to manufacturer’s instructions and was used for bulk RNA-seq. Illumina-compatible libraries were prepared according to manufacturer’s instructions and next generation sequencing (NGS) (paired-end and strand-specific) was performed on an Illumina HiSeq2000. For scRNA-seq, approximately 16.000 sorted G+ cells were loaded in a channel of a chromium controller (10× Genomics) for generation of gel-bead-in-emulsions (Greek Research Infrastructure for Personalized Medicine, pMedGR). The sequencing library was prepared using Single Cell 3′ Reagent Kits v3.1 (10× Genomics) and sequenced on an Illumina NextSeq2000.
Bioinformatic analysis for both RNA-seq and scRNA-seq data is described in detail in theSupplementary Data
Bioinformatic analysis for both RNA-seq and scRNA-seq data is described in detail in the
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