Cells were sorted as described in “Tissue Preparation and Flow Cytometry” and immediately lysed with RLT Buffer from the Qiagen RNeasy Plus Mini kit or RNA extraction buffer from PicoPure RNA isolation kit (Arcturus Bioscience, Inc.). Cell lysates were stored at −80°C until RNA was extracted. RNA isolations were performed using the Qiagen RNeasy Plus Mini kit or PicoPure RNA isolation kit. RNA quality and quantity were measured using the Agilent Technologies High Sensitivity RNA ScreenTape System. SMART-Seq v4 Ultra Low Input RNA kit (Clontech Laboratories, Inc.) was used for full-length cDNA synthesis, and the Nextera XT DNA sample preparation kit (Illumina Inc.) was used for library preparation. DNA libraries were sequenced on an Illumina NextSeq 500 instrument with a target read depth of ∼10 million aligned reads per sample. The pool was denatured and diluted, resulting in a 2.5 pM DNA solution. PhiX control was spiked at 1%, and the pool was sequenced by 1 × 75 cycles using the NextSeq 500 High Output reagent kit (Illumina Inc.).
Nextseq 500 high output reagent kit
Manufactured by Illumina
The NextSeq 500 High Output reagent kit is a laboratory consumable product designed for use with the NextSeq 500 sequencing system. The kit contains the necessary reagents and materials to perform sequencing runs on the instrument.
Automatically generated - may contain errors
Lab products found in correlation
Nextseq 500 instrument, by Illumina (4 mentions)
Rneasy plus mini kit, by Qiagen (2 mentions)
Truseq nano dna sample preparation kit, by Illumina (2 mentions)
Nextera dna sample preparation kit, by Illumina (2 mentions)
Smart seq v4 ultra low input rna kit, by Takara Bio (1 mentions)
Picopure rna isolation kit, by Arcturus (1 mentions)
Nextera xt dna sample preparation kit, by Illumina (1 mentions)
Picopure rna isolation kit, by Qiagen (1 mentions)
High sensitivity rna screen tape system, by Agilent Technologies (1 mentions)
Bioanalyzer, by Agilent Technologies (1 mentions)
Agilent 4200 tapestation, by Agilent Technologies (1 mentions)
Oligo dt beads, by New England Biolabs (1 mentions)
Rnaeasy plus mini kit, by Qiagen (1 mentions)
Nebnext ultra rna kit, by New England Biolabs (1 mentions)
Nextseq 500, by Illumina (1 mentions)
8 protocols using nextseq 500 high output reagent kit
1
RNA-Seq Library Preparation from Sorted Cells
2
Optimized Library Preparation for Illumina Sequencing
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A quality analysis (Bioanalyzer, Agilent, Santa Clara, CA, USA) was
performed to check DNA amplicon length prior to library preparation. A total of
255 libraries (242 samples, 9 PCR negative controls with Sigma water and 4 PCR
negative controls containing human DNA) were prepared using the TruSeq Nano DNA
Sample Preparation kit according to the user guide revision A (Illumina, San
Diego, CA, USA) with the following modifications: as each sample consisted of
approximately 200–400 bp long PCR products, the tagmentation, end-repair and size
selection steps were omitted, and hence the library preparation started with
adenylation of 3’-ends. We used 75 ng of PCR product as input in a volume of
17.5 μl of resuspension buffer and 2 adaptor indexed primers were ligated to each
sample.
All individual libraries were validated, normalised to 2 nM and
pooled in different pools. Each pool contained approximately 48 libraries
(including specimens and negative controls) and was denatured and diluted,
resulting in a 1.8 pM DNA solution. All library pools were sequenced paired-end
151 + 151 cycles once, using the NextSeq 500 instrument and NextSeq 500 High
Output reagent kit (Illumina) as described in the user guides Denature and Dilute
Libraries Guide v02 for the NextSeq System, NextSeq 500 kit Reference Guide
revision F and NextSeq 500 System Guide v02.
performed to check DNA amplicon length prior to library preparation. A total of
255 libraries (242 samples, 9 PCR negative controls with Sigma water and 4 PCR
negative controls containing human DNA) were prepared using the TruSeq Nano DNA
Sample Preparation kit according to the user guide revision A (Illumina, San
Diego, CA, USA) with the following modifications: as each sample consisted of
approximately 200–400 bp long PCR products, the tagmentation, end-repair and size
selection steps were omitted, and hence the library preparation started with
adenylation of 3’-ends. We used 75 ng of PCR product as input in a volume of
17.5 μl of resuspension buffer and 2 adaptor indexed primers were ligated to each
sample.
All individual libraries were validated, normalised to 2 nM and
pooled in different pools. Each pool contained approximately 48 libraries
(including specimens and negative controls) and was denatured and diluted,
resulting in a 1.8 pM DNA solution. All library pools were sequenced paired-end
151 + 151 cycles once, using the NextSeq 500 instrument and NextSeq 500 High
Output reagent kit (Illumina) as described in the user guides Denature and Dilute
Libraries Guide v02 for the NextSeq System, NextSeq 500 kit Reference Guide
revision F and NextSeq 500 System Guide v02.
3
Illumina NextSeq library preparation
4
RNA-seq Analysis of Epithelial Cells
5
Nextera DNA Library Preparation
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50 ng of genomic DNA per sample was tagmented with 2 unique indices, as described (Nextera DNA Sample Preparation kit, user guide revision B (Illumina)). The individual libraries were validated, normalized to 4 nM, pooled, denatured and diluted, resulting in a 2.6 pM DNA solution, which was spiked at 1% PhiX control and the library pool was sequenced paired-end 151+151 cycles using NextSeq 500 High Output reagent kit (Illumina) as described in the user guides “Denaturing and Diluting Libraries for the NextSeq 500” revision A and “NextSeq 500 kit Reference Guide” revision F.
6
RNA Isolation and RNA-seq Library Preparation
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RNA from cell lines was isolated using the RNAeasy Plus mini kit (Qiagen, Germany). RNA quality and quantity were measured using Agilent 4,200 Tapestation with the high Sensitivity RNA ScreenTape System (Agilent Technologies). Briefly, mRNA was isolated from 80 ng of purified total RNA using oligo-dT beads (New England Biolabs, Inc). An NEBNext Ultra™ RNA kit (New England Biolabs, United States) was used for full-length cDNA synthesis and library preparation. Libraries were pooled, denatured and diluted, resulting in a 2.0 p.m. DNA solution. PhiX control was spiked at 1%. Libraries were sequenced on an Illumina NextSeq 500 instrument (Illumina Inc) using NextSeq 500 High Output reagent kit (Illumina Inc) (1x75 cycles) with a target read depth of approximate (5–10) million aligned reads per sample.
7
Nextera-based DNA library preparation
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50 ng of WGA genomic DNA per sample (n = 95) was used for DNA library preparation with a dual index system using the Nextera DNA Sample Preparation kit according to the user guide revision B (Illumina). The individual libraries were validated and normalized to 4 nM. Libraries were then divided in 4 sets, each set containing at least 3 negative controls (1 water control and 2 blank blocks) and 20 NMSC specimens. Each set was denatured and diluted, resulting in a 1.8 pM DNA solution, and finally sequenced by paired-end 151+151 cycles using NextSeq 500 High Output reagent kit (Illumina). The sequencing preparations were made according to the user guides, Denaturing and Diluting Libraries for the NextSeq500 revision A and NextSeq500 kit Reference Guide revision F.
8
RNA-seq Transcriptome Analysis Pipeline
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Libraries were sequenced on an Illumina NextSeq 500 instrument (Illumina Inc) using NextSeq 500 High Output reagent kit (Illumina Inc) (1 × 75 cycles) with a target read depth of approximately (5–10) million aligned reads per sample. FASTQ files were generated using bcl2fastq 2.19.1 (Illumina). To facilitate reproducible analysis, samples were processed using the publicly available nf-core/RNA-seq pipeline version 3.4 implemented using nextflow 21.10.5.5658 and singularity 3.8.12 with the public nu_genomics configuration. The STAR/Salmon method was used for transcriptome alignment and gene-level count assignment. The GRCm38 genome (Ensembl release 81) was used as a reference. The resulting length-scaled DESeq2 object was then imported directly for downstream analysis.
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