Single-cell RNAseq was performed on the 10X Chromium platform and libraries prepared at the MRC WIMM Institute Single Cell Core Facility using the Chromium Single Cell 3’ Library and Gel Bead Kit v2, 4 rxns PN-120267. E2:Citrine was electroporated in ovo into the lumen of the neural tube and incubated until HH18 stage. The vagal region from somites 1-7 axial level was dissected and dissociated as described above, prior to FAC-sorting. Approximately 2000 FAC-sorted cells were pooled with a collaborator’s experiment using zebrafish cells (carrying mCherry transgene). Final libraries were sequenced on the standalone mode on the Illumina NextSeq 500 platform using TG NextSeq® 500/550 High Output Kit v2 (150 cycles) and settings set to paired-end single index parameters as specified in the manufacturer’s protocol.
Tg nextseq 500 550 high output kit v2
Manufactured by Illumina
The TG NextSeq 500/550 High Output Kit v2 is a sequencing reagent kit designed for use with the Illumina NextSeq 500 and NextSeq 550 sequencing systems. The kit provides the necessary reagents for performing high-throughput DNA or RNA sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Nextseq 500 platform, by Illumina (4 mentions)
Bbcl2fastq software, by Illumina (3 mentions)
2200 tapestation instrument, by Agilent Technologies (3 mentions)
Ultraclean microbial dna isolation kit, by Qiagen (3 mentions)
Nextseq sequencer, by Illumina (3 mentions)
Nebnext ultra 2 dna library prep kit, by Illumina (3 mentions)
Ambion rnaqueous micro total rna isolation kit, by Thermo Fisher Scientific (2 mentions)
Takara smart seq v4 ultra low input rna kit, by Takara Bio (2 mentions)
Tapestation 2200, by Agilent Technologies (2 mentions)
Nextseq 550, by Illumina (2 mentions)
Nebnext ultra 2 dna library prep kit for, by Illumina (2 mentions)
Smarter universal low input rna kit, by Takara Bio (1 mentions)
Qubit cdna hs assay kits, by Thermo Fisher Scientific (1 mentions)
Smarter thruplex dna seq kit, by Takara Bio (1 mentions)
Nextseq platform, by Illumina (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
M220 focused ultrasonicator, by Covaris (1 mentions)
High sensitivity rna screentape, by Agilent Technologies (1 mentions)
Dna lobind tube, by Eppendorf (1 mentions)
14 protocols using tg nextseq 500 550 high output kit v2
1
Single-cell RNAseq of Avian Neural Tube
2
RNA-Seq Library Preparation from FACS-Sorted Fibroblasts
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RNA isolation and cDNA preparation of FACS-sorted fibroblasts were performed using the SMARTer® Universal Low Input RNA Kit (Takara Bio, Saint-Germain-en-Laye, France) according to the manufacturer’s instructions. Prepared cDNA was purified by immobilization on AMPure XP beads (Beckman Coulter), and quantified using Qubit™ cDNA HS Assay Kits (ThermoFisher Scientific, Dreieich, Germany). Quality of purified cDNA was checked using the Agilent 2100 Bioanalyzer® with High Sensitivity DNA Chips. Purified cDNA of sufficient quality was sheared using a M220 focused ultrasonicator (Covaris, Brighton, UK), yielding cDNA fragments around 400 bp. Fragmented cDNA was then used to prepare libraries using the SMARTer ThruPLEX DNA-Seq Kit (Takara Bio) according to the manufacturer’s instructions. Amplified libraries were purified by immobilization on AMPure XP beads, and quality and DNA content were checked using High Sensitivity DNA Chips as well as again with Qubit™ cDNA HS Assay Kits. Libraries were diluted, denatured according to Illumina Denture and Dilute Libraries Guide, and mixed with PhiX control (8%). Six to eight libraries were loaded on one sequencing cartridge of the TG NextSeq® 500/550 High Output Kit v2 (75 cycles) (Illumina, Eindhoven, The Netherlands) and sequencing was performed on the NextSeq platform (Illumina).
3
Bulk RNA-seq of FAC-sorted Avian Cells
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Total RNAs from FAC-sorted cells were isolated using the Ambion RNAqueous Micro Total RNA isolation kit (Cat #AM1931, ThermoFisher Scientific) and integrity checked using Agilent Tapestation (only samples with a RNA Integrity Number >6 were used for library preparations). Samples were stored at -80ºC until all replicates were collected and sample libraries were prepared on the same day to prevent batch effects. Libraries were prepared using Takara SMART-Seq™ v4 Ultra™ Low Input RNA kit (Cat #634889, Takara Bio Clontech) and sequenced using 40 bp paired-end reads on the Illumina NextSeq 500 platform using TG NextSeq® 500/550 High Output Kit v2 (75 cycles). Two biological replicates were used for each stage (HH10, HH18. HH25) and sample (double positive, single E2-only and Citrine-negative control per stage).
4
Probiotic Capsule Metagenome DNA Extraction
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Total metagenome DNA of the probiotic capsule sample was extracted using the UltraClean Microbial DNA Isolation kit from MoBio Laboratories. The quality of the isolated total metagenome DNA was checked using an Agilent Tapestation 2200 instrument. The DNA sample was used for in vitro fragment library preparation. In vitro fragment library way prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina. Paired-end fragment reads were generated on an Illumina NextSeq sequencer using TG NextSeq 500/550 High Output Kit v2 (300 cycles). Primary data analysis (base-calling) was carried out with Bbcl2fastq software (v2.17.1.14, Illumina).
5
RNA Extraction and Sequencing of Single Cells
6
Metagenomic Analysis of Kefir and Yogurt
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Total metagenome DNA of kefir (k_g_04) and yoghurt (y_g_01) samples were extracted using the UltraClean Microbial DNA Isolation kit from MoBio Laboratories. The quality of the isolated total metagenomic DNA was checked using an Agilent Tapestation 2200 instrument. The DNA samples were used for in vitro fragment library preparation. In vitro fragment libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina. Paired-end fragment reads were generated on an Illumina NextSeq sequencer using TG NextSeq 500/550 High Output Kit v2 (300 cycles). Read numbers were the following: 22,044,496 and 20,895,112 for kefir and yoghurt, respectively. Primary data analysis (base-calling) was carried out with Bbcl2fastq software (v2.17.1.14, Illumina).
7
Single-cell RNAseq of Avian Neural Tube
8
RNA Extraction and RNA-seq Analysis of OHCs
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RNA was obtained from pools of three OHCs, from three different animals, cultured with or without B1 for 9 days. Total RNA was extracted using miRNeasy Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. All samples had their RNA quantified by fluorometry using Qubit RNA HS Assay Kit (Invitrogen, Carlsbad, CA) with Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) and had their quality assessed by capillary electrophoresis with a Bioanalyzer 2100 (Agilent, Santa Clara, CA).
Later, libraries were produced using the TruSeq Stranded mRNA Kit (Illumina, San Diego, CA) and the indexed fragments were sequenced using the TG NextSeq 500/550 High Output Kit v2 (Illumina, San Diego, CA).
Later, libraries were produced using the TruSeq Stranded mRNA Kit (Illumina, San Diego, CA) and the indexed fragments were sequenced using the TG NextSeq 500/550 High Output Kit v2 (Illumina, San Diego, CA).
9
Bulk RNA-seq of FAC-sorted Avian Cells
10
Target Capture and Paired-End Sequencing
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Target capture was performed according to the manufacturer’s protocol using the SureSelect XT automation reagent kit (Agilent, Santa Clara, CA), and a paired-end sequencing library was prepared using a barcode. The size and quality of the genomic DNA were validated using the Genomic DNA Analysis ScreenTape and Genomic DNA Reagent together with the Agilent 4200 Tape station (Agilent). Libraries were sequenced using the TG NextSeq 500/550 High Output Kit v2 (Illumina, Inc., San Diego, CA) and TG NextSeq 500/550 Mid Output Kit v2 (Illumina, Inc.).
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