Colonic LP immune cells were isolated and sorted for live CD45+ cells by FACS. Cells were resuspended at a concentration of 700–1,200 cells/μl for microfluidics (Chromium Single-Cell Controller; 10x Genomics). Cells were loaded into the chip and run using the Chromium Single Cell 3′ Reagent Kit v3 (10x Genomics) according to the manufacturer’s instructions. Resuspended single cells were partitioned in gel beads in emulsion and lysed. Lysis was followed by RNA barcoding, reverse transcription, PCR amplification (12–14 cycles), fragmentation, ligation, and sample index PCR. cDNAs were quantified by D5000 ScreenTape (Agilent Technologies) on an Agilent 4200 TapeStation system (Agilent Technologies). Sequencing-ready scRNAseq libraries were quantified by 2100 Bioanalyzer (Agilent Genomics) instrument. Sequencing was performed on an Illumina NextSeq 500 machine (San Diego), and four indexed samples were multiplexed into one output flow cell using NextSeq 500/550 High Output Kit v2.5 in paired-end sequencing (R1, 26nt; R2, 98nt, and i7 index 8nt) at the MD Anderson Cancer Center South Campus RNAseq core facility.
Nextseq 500 machine
Manufactured by Illumina
Sourced in United States
The NextSeq 500 is a desktop sequencing system designed for a wide range of applications, including gene expression analysis, target enrichment, and small genome sequencing. The instrument utilizes Illumina's proprietary sequencing-by-synthesis technology to generate high-quality sequencing data efficiently.
Automatically generated - may contain errors
Lab products found in correlation
2100 bioanalyzer, by Agilent Technologies (5 mentions)
Tapestation 4200, by Agilent Technologies (5 mentions)
Nextseq 500 550 high output kit v2, by Illumina (4 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (4 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (4 mentions)
Ampure xp bead, by Beckman Coulter (3 mentions)
Single cell rna seq kit, by 10x Genomics (3 mentions)
Chromium next gem single cell 3 reagent kits v3, by 10x Genomics (3 mentions)
Chromium system, by 10x Genomics (3 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Nextera xt dna library preparation kit, by Illumina (2 mentions)
Clontech smart seq ht kit, by Takara Bio (2 mentions)
Rneasy plus micro kit, by Qiagen (2 mentions)
Mirneasy micro kit, by Qiagen (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (2 mentions)
Phusion hot start flex dna polymerase, by New England Biolabs (2 mentions)
Tagment dna buffer 2x reaction buffer, by Illumina (2 mentions)
Transposition mix and purification, by Illumina (2 mentions)
Minelute pcr purification kit, by Qiagen (2 mentions)
67 protocols using nextseq 500 machine
1
Single-Cell RNA Sequencing of Colonic Immune Cells
2
CM Metagenomic DNA Extraction and Sequencing
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Genomic DNA (gDNA) from 25 randomly selected CM samples was extracted by an automated Maxwell 16 DNA extraction platform using blood DNA purification kits (Promega, United Kingdom) following previously described protocols (Hoque et al., 2019 (link)). DNA quantity and purity were determined with NanoDrop (ThermoFisher, United States) by measuring 260/280 absorbance ratios. Sequencing libraries were prepared with the Nextera XT DNA Library Preparation Kit (Head et al., 2014 (link)) and paired-end (2 × 150 bp) sequencing was performed on a NextSeq 500 machine (Illumina Inc., United States) at the George Washington University Genomics Core facility. Our metagenomic DNA yielded a total of 596.74 million reads with an average of 23.87 million (maximum = 39.75 million, minimum = 8.89 million) reads per sample (Supplementary Material ) before cleaning.
3
Exosomal RNA Isolation and Sequencing
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For RNA isolation, exosomes were prepared using PEG precipitation, resuspended in cold PBS and centrifuged in Optima XPN-100 Ultracentrifuge (Beckman Coulter) at 100,000 x g, 4°C for 75 min. After ultracentrifugation, pellets were resuspended in 200 μl of ice cold Exosome Resuspension Buffer (Total Exosome RNA & Protein Isolation Kit, Invitrogen) and stored at -80°C. RNA was isolated using the manufacturer’s protocol. For preparation of a library of RNAs longer than 200 nucleotides, TruSeq Stranded mRNA kit (Illumina) was used. Library of miRNAs was prepared using the TruSeq small RNA kit (Illumina). Both libraries were sequenced on the Illumina NextSeq 500 machine. Single-end reads of 75 bp were sequenced for the long RNAs and 50 bp single-end reads—for the small RNAs.
4
ChIP-exo Assay for Transcription Factors
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Approximately 100 million HEK293T cells were crosslinked for each ChIP-exo assay using the ChIP-seq protocol described above. Crosslinked cells, ZFX antibody (Cell Signaling Technology # 5419S), and ZNF711 antibody (Thermo Fisher #PA5-31815) were sent to Peconic, where the ChIP-exo assay was performed (http://www.peconicgenomics.com/services.html ). Samples were sequenced on an Illumina NextSeq 500 machine using 2 × 40 bp paired-end sequencing generating ∼40 million reads per sample. Sequence reads were aligned to human (hg19) genome using using bwa-mem (v0.7.9a) (http://bio-bwa.sourceforge.net/ ). Peaks in ChIP-exo data were called using ChExMix (http://mahonylab.org/software/chexmix/ ).
5
Automated Chromosomal Aneuploidy Detection
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Sequencing was performed using a NextSeq 500 machine (Illumina; up to 77 or 68 cycles – single end; excluding sample-specific barcodes). Reads were afterwards aligned to the human reference genome (GRCh38/hg38) using Bowtie2 (version 2.2.4 or 2.3.4.169 (link);). Duplicate reads were marked with BamUtil (version 1.0.3;70 (link)) or Samtools markdup (version 1.971 (link). The aligned read data (bam files) were analyzed with a copy number calling algorithm called AneuFinder [https://github.com/ataudt/aneufinder ]72 (link). Following GC correction and blacklisting of artefact-prone regions (extreme low or high coverage in control samples), libraries were analyzed using the dnacopy and edivisive copy number calling algorithms with variable width bins (average bin size = 1 Mb; step size = 500 kb). Results were afterwards curated by requiring a minimum concordance of 95% between the results of the two algorithms. Libraries with on average less than 10 reads per bin (~ 55,000 reads for a diploid genome) were discarded. A chromosome was classified as aneuploid when at least 95 % of the bins showed a deviation from euploid (deviation from 2-somy). Chromosomes 10 and 12 were excluded for the calculation of whole-genome scores.
6
Transcriptome and Methylome Profiling
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Both gonads and liver samples were sent to the DeepSeq sequencing facility at Nord University (Bodø, Norway) for RNA-sequencing (RNA-seq) where libraries were prepared using an NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs). The libraries were subsequently sequenced by the NextSeq500 machine (Illumina). See Additional file 1 : RNA-seq library preparation and sequencing in Supplementary Methods for further details.
Both gonads and liver samples were sent to the CeMM Biomedical Sequencing Facility (Vienna, Austria) for reduced representation bisulfite sequencing (RRBS) where enzyme digestion by MspI and TaqI were performed followed by size selection and bisulfite conversion. RRBS was subsequently performed using the HiSeq 3000/4000 instruments (Illumina). See Additional file1 : RRBS library preparation and sequencing in Supplementary Methods for further details.
Both gonads and liver samples were sent to the CeMM Biomedical Sequencing Facility (Vienna, Austria) for reduced representation bisulfite sequencing (RRBS) where enzyme digestion by MspI and TaqI were performed followed by size selection and bisulfite conversion. RRBS was subsequently performed using the HiSeq 3000/4000 instruments (Illumina). See Additional file
7
Droplet-based 3' end scRNA-Seq
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Droplet-based digital 3′ end scRNA-Seq was performed on a Chromium Single-Cell Controller (10 × Genomics, Pleasanton, CA) using the Chromium Single Cell 3′ Reagent Kit v2 according to the manufacturer’s instructions. Briefly, suspended single cells were partitioned in Gel Beads in Emulsion (GEMs) and lysed, followed by RNA barcoding, reverse transcription and PCR amplification (12–14 cycles). Sequencing-ready scRNA-Seq were prepared according to the manufacturer’s instructions, checked and quantified on 2100 Bioanalyzer (Agilent Genomics, Santa Clara, CA) and Qubit 3.0 (Invitrogen, Carlsbad, CA) instruments. Sequenced was performed on a NextSeq 500 machine (Illumina, San Diego, CA) using the NextSeq 500/550 High Output v2 kit (75 cycles).
8
RNA-seq Library Preparation from Cells
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Total RNAs from scramble shRNA or TDP-43 shRNA treated cells were used to construct RNA-sequencing libraries using the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian kit (Takara 634411), according to the manufacturer’s instructions. The resulting libraries were quantitated, pooled, and sequenced on a Nextseq 500 machine using the 150-cycle high output kit in a 75bp paired-end mode (Illumina).
9
Simultaneous scRNA-seq and scATAC-seq Library Generation
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Immediately following dissociation, cells were diluted to 1200 cells/µL in preparation for scRNA-seq and scATAC-seq library generation as described in our previous work [37 (link)]. For scRNA-seq libraries, 10,000 cells were used for library preparation using the 10× Genomics Single Cell 3′ kits: Single Cell 3′ GEM, Library & Gel Bead Kit v3 (PN-1000075), Chromium Chip B Single Cell Kit (PN-10000153), and Chromium i7 Multiplex Kit (PN-120262) following the manufacturer’s protocol.
For scATAC-seq, nuclei isolation of 500,000 cells was carried out following the Nuclei Isolation for Single Cell ATAC Sequencing protocol from 10× Genomics using a four-minute lysis time. Next, 10,000 nuclei were used for library preparation using the 10× Genomics Single Cell ATAC Kits: Chromium Single Cell ATAC Library & Gel Bead Kit v1 (PN-1000110), Chromium Chip E Single Cell ATAC Kit (PN-1000082), and Chromium i7 Multiplex Kit N, Set A (PN-1000084) following the manufacturer’s protocol. scRNA-seq and scATAC-seq libraries were sequenced using 10× Genomics’ suggested sequencing parameters on an Illumina NextSeq 500 machine by UNC’s Translational Genomics Lab.
For scATAC-seq, nuclei isolation of 500,000 cells was carried out following the Nuclei Isolation for Single Cell ATAC Sequencing protocol from 10× Genomics using a four-minute lysis time. Next, 10,000 nuclei were used for library preparation using the 10× Genomics Single Cell ATAC Kits: Chromium Single Cell ATAC Library & Gel Bead Kit v1 (PN-1000110), Chromium Chip E Single Cell ATAC Kit (PN-1000082), and Chromium i7 Multiplex Kit N, Set A (PN-1000084) following the manufacturer’s protocol. scRNA-seq and scATAC-seq libraries were sequenced using 10× Genomics’ suggested sequencing parameters on an Illumina NextSeq 500 machine by UNC’s Translational Genomics Lab.
10
Transcriptome Sequencing of AdrKO Mice
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Transcriptome deep sequencing (RNA-seq) was performed using total RNA isolated from lung tissue of three AdrKO and three age-matched littermates (male F2 intercross mice). Three individuals from each genotypic group were randomly selected. Total RNA was extracted from frozen tissue using the SV Total RNA Isolation System (Promega Corporation, Madison, WI) according to the manufacturer's instructions. The quantity and quality of RNA samples were assessed by Nanodrop 1000 (Thermo Fisher Scientific Inc., Wilmington, DE, USA). Total RNA samples were sent to DRIGEN Co., Ltd. for RNA-seq library preparation using the TruSeq SBS Kit (75 Cycles) and single end sequencing by means of an Illumina NextSeq 500 machine (Illumina). RNA-seq reads were quality filtered using SolexaQA packages with default parameters and a filter for the requisite length greater than 70 bp for both ends of each read pair. Sequencing data have been submitted to the NCBI Sequence Read Archive.
Quality filtered RNA-seq reads were mapped to the mouse reference genome, mm10, with TopHat v2.1.0. The comparisons between treated and normal mice were made using custom Perl scripts. Genes that showed significant (P < 0.05) difference in transcript levels were termed as differentially expressed (DE) genes.
Quality filtered RNA-seq reads were mapped to the mouse reference genome, mm10, with TopHat v2.1.0. The comparisons between treated and normal mice were made using custom Perl scripts. Genes that showed significant (P < 0.05) difference in transcript levels were termed as differentially expressed (DE) genes.
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