For long RNA library preparation a Ribo-zero Magnetic Gold rRNA removal kit (Epicenter, IIlumina Inc.) was used to remove ribosomal RNA from the total RNA. First strand synthesis was performed using NEBNext RNA first strand synthesis module (New England BioLabs Inc., Ipswich, MA, USA). Immediately thereafter, directional second strand synthesis was performed using NEBNExt Ultra Directional second strand synthesis kit. Following this, cDNAs were used for standard library preparation protocol using NEBNext DNA Library Prep Master Mix Set for Illumina. End-repair was performed followed by polyA addition and custom adapter ligation. After ligation, material was individually barcoded with unique in-house genomics service lab primers. Library quality and concentration were assessed by a Qubit 2.0 Fluorometer and DNA 1000 chips on an Agilent 2100 Bioanalyzer. Accurate quantification for sequencing applications was determined using the qPCR-based KAPA Biosystems Library Quantification kit (Kapa Biosystems, Inc., Woburn, MA). Each library was diluted to a final concentration of 12.5 nM and pooled equimolar prior to clustering. Paired-end sequencing was performed on all samples (100 bp paired-end directional reads). Raw reads were de-multiplexed using a bcl2fastq conversion software v1.8.3 (Illumina, Inc.) with default settings.
Bcl2fastq conversion software v1
Manufactured by Illumina
Sourced in United States
Bcl2fastq Conversion Software v1.8.4 is a bioinformatics tool that converts Illumina's proprietary BCL file format into the more widely used FASTQ format. This software enables users to process raw sequencing data generated by Illumina's sequencing platforms.
Automatically generated - may contain errors
Lab products found in correlation
Real time analysis v1, by Illumina (11 mentions)
Hiseq 2500, by Illumina (8 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (3 mentions)
Sciclone ngsx workstation, by PerkinElmer (3 mentions)
Agilent 2200 tapestation, by Agilent Technologies (3 mentions)
Qubit 2.0 fluorometer, by Agilent Technologies (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Truseq rna sample prep kit, by Illumina (2 mentions)
Kapa biosystems library quantification kit, by Roche (1 mentions)
Nebnext rna first strand synthesis module, by New England Biolabs (1 mentions)
Nebnext dna library prep master mix set, by Illumina (1 mentions)
Hiseqh2000, by Illumina (1 mentions)
Agilent 2100 bioanalyzer, by Thermo Fisher Scientific (1 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Nebnext mrna library prep reagent set for, by Illumina (1 mentions)
Maxwell 16 lev simplyrna kit, by Promega (1 mentions)
Nebnext magnetic oligo d t 25 beads, by New England Biolabs (1 mentions)
Hiseq 2000 sequencer, by Illumina (1 mentions)
Nebnext ultratm 2 dna library prep kit for, by Illumina (1 mentions)
Hiseq system, by Illumina (1 mentions)
25 protocols using bcl2fastq conversion software v1
1
Directional RNA Sequencing Library Prep
2
RNA Sequencing Workflow for Differential Gene Expression
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All RNA samples were subjected to RNA sequencing (RNAseq; HudsonAlpha Genomic Services Lab, Huntsville, AL) as performed before [37 (link)]. In short, total RNA concentration was estimated by Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) and RNA integrity by using the Agilent 2100 Bioanalyzer (Applied Biosystems, Carlsbad, CA, USA). RNAseq libraries were formed from approximately 500 ng total RNA of each pooled sample, followed by poly(A) enrichment. RNAseq was performed using paired-end sequencing on Illumina HiSeqH2000 (Illumina, San Diego, CA, USA), at 50 base pairs, generating over 25 million paired reads per sample. Raw RNAseq FASTQ files were demultiplexed by bcl2fastq conversion software v1.8.3 (Illumina, Inc., San Diego, CA, USA) using default settings.
RNAseq data was analyzed using GeneSifter software (VizX Labs, Seattle, WA). RNAseq reads were mapped to the Mus musculus reference genome build 37.2, and for this, the reads were trimmed by 15 base pairs at the five-prime end. Subsequently, transcript abundance was calculated by estimating the reads per kilobase of exon per million mapped reads (RPKM), and normalization to the number of mapped reads was used for comparison of two mRNA sets. A t test was used for pairwise comparison and a likelihood ratio test to adjust for distribution probability.
RNAseq data was analyzed using GeneSifter software (VizX Labs, Seattle, WA). RNAseq reads were mapped to the Mus musculus reference genome build 37.2, and for this, the reads were trimmed by 15 base pairs at the five-prime end. Subsequently, transcript abundance was calculated by estimating the reads per kilobase of exon per million mapped reads (RPKM), and normalization to the number of mapped reads was used for comparison of two mRNA sets. A t test was used for pairwise comparison and a likelihood ratio test to adjust for distribution probability.
3
RNA-seq Analysis of KDM5B Knockout
4
RNA-Seq Library Preparation and Analysis
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RNA-Seq experiments were performed as previously described [29 (link)]. Briefly, RNA was extracted from sorted cells using the automated Maxwell 16 magnetic particle processor and a Maxwell 16 LEV simply RNA kit (Promega). Polyadenylated RNAs were isolated using NEBNext magnetic oligo d(T)25 beads (New England BioLabs), individually bar-coded, and next generation sequencing expression libraries were prepared using the NEBNext mRNA Library Prep Reagent Set for Illumina (New England BioLabs). Each library was diluted to a final concentration of 12.5nM and pooled equimolar prior to clustering. Paired-End (PE) sequencing (25 million, 50-bp, paired-end reads) was performed using a 200 cycle TruSeq SBS HS v3 kit (Illumina) on an Illumina HiSeq2000 sequencer. Image analysis and base calling were performed using the standard Illumina Pipeline consisting of Real time Analysis (RTA) v1.13. Raw reads were de-multiplexed using a bcl2fastq conversion software v1.8.3 (Illumina) with default settings. Paired-end reads were mapped against the human reference genome (GRCh37) using TopHat v2.0.0. Human gene models and annotations were obtained from ENSEMBL v63 (June 2011). Gene expression quantification was conducted on the gene level using Subread v1.4.6 counting mapped paired-reads to obtain fragment counts per gene (see S2 Text
5
High-throughput Metagenomics Sequencing
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High-throughput sequencing of the samples was performed by Genome Quebec using the HiSeq system (Illumina). Libraries were prepared using NEBNext UltraTM II DNA Library Prep Kit for Illumina (New England Biolabs) followed by sequencing on two lanes of an Illumina HiSeq 4000 PE150 system (Illumina) allocating 1/20 and 1/25 of a lane for each sample. Demultiplexing and conversion to FASTQ files were performed using bcl2fastq Conversion Software v1.8.4 (Illumina) resulting in 32 metagenomic datasets.
6
Whole Exome Sequencing of Frozen Tissue
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Genomic DNA from frozen tissue was extracted using the Qiagen Gentra Puregene Tissue Kit (Qiagen). Sequencing libraries were prepped with the KAPA HyperPrep Kit (Roche) using 1 µg of DNA. DNA was sheared using a Covaris LE220 ultrasonicator targeting 200 bp, and sequencing adaptors added by ligation. Individually barcoded libraries were pooled 4-plex before capture. Libraries were hybridized to SeqCap EZ Choice probes of the 50 Mb Human UTR Design (Roche), and sequenced on a HiSeq 2500 (Illumina) using a PE100 in high-output mode. Image analysis and base calling were performed using Illumina’s Real Time Analysis v1.18.66.3 software, followed by demultiplexing of indexed reads and generation of FASTQ files, using Illumina’s bcl2fastq Conversion software v1.8.4.
7
RNA-seq Library Preparation and Sequencing
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RNA was extracted and purified from the Ptm lymphocytes using the RNeasy Mini kit (Qiagen) following manufacturer's protocol. Total RNA integrity was checked using an Agilent 2200 TapeStation (Agilent Technologies) and quantified using a Trinean DropSense96 spectrophotometer (Caliper Life Sciences). RNA-seq libraries were prepared from total RNA using the TruSeq RNA Sample Prep Kit v2 (Illumina) and a Sciclone NGSx Workstation (PerkinElmer). Library size distributions were validated using an Agilent 2200 TapeStation (Agilent Technologies). Additional library QC, blending of pooled indexed libraries, and cluster optimization were performed using Life Technologies’ Invitrogen Qubit 2.0 Fluorometer (Life Technologies-Invitrogen). RNA-seq libraries were pooled (6-plex) onto a flow cell lane. Sequencing was performed using an Illumina HiSeq 2500 in rapid mode employing a paired-end, 50 base read length (PE50) sequencing strategy. Image analysis and base calling were performed using Illumina's Real Time Analysis v1.18 software, followed by 'demultiplexing' of indexed reads and generation of FASTQ files, using Illumina's bcl2fastq Conversion Software v1.8.4.
8
Prokaryotic RNA-Seq Library Preparation
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RNA was processed using the Ovation Complete Prokaryotic RNA-Seq Library System
(NuGEN, #0363–32, 0326–32, 0327–32) according to the manufacturer’s instructions
to a final pooled library concentration of 3 nM. Libraries were sequenced on an
Illumina HiSeq 2500 (SR50) at The Genomics Resource at the Fred Hutchinson
Cancer Research Center. Image analysis and base calling were performed using
Illumina's Real Time Analysis v1.18.66.3 software, followed by 'demultiplexing'
of indexed reads and generation of FASTQ files using Illumina's bcl2fastq
Conversion Software v1.8.4. Reads determined by the RTA software to pass
Illumina's default quality filters were concatenated for further analysis. The
FASTQ files were aligned and analyzed using Rockhopper software (McClure et al., 2013 (link)). These data have
been deposited to the GEO and are accessible using accession number
GSE150188.
(NuGEN, #0363–32, 0326–32, 0327–32) according to the manufacturer’s instructions
to a final pooled library concentration of 3 nM. Libraries were sequenced on an
Illumina HiSeq 2500 (SR50) at The Genomics Resource at the Fred Hutchinson
Cancer Research Center. Image analysis and base calling were performed using
Illumina's Real Time Analysis v1.18.66.3 software, followed by 'demultiplexing'
of indexed reads and generation of FASTQ files using Illumina's bcl2fastq
Conversion Software v1.8.4. Reads determined by the RTA software to pass
Illumina's default quality filters were concatenated for further analysis. The
FASTQ files were aligned and analyzed using Rockhopper software (McClure et al., 2013 (link)). These data have
been deposited to the GEO and are accessible using accession number
GSE150188.
9
RNA-seq Data Analysis Workflow
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Demultiplexing of the RNA-seq samples was performed with the bcl2fastq Conversion software v1.8.4 (Illumina). Next, the quality check, trimming of reads, and mapping of reads were conducted with FastQC 11.8, FLEXBAR 3.5.0, and TopHat 2.1.1 software, respectively. The mapping statistics and read counts were generated with samtools 1.9, RSeQC, and HTSeq-count 0.11.1 software, Gtf-Ensembl annotation 96. Differential expression analysis was performed using DEseq 2 software. Genes with p-adjusted < 0.05 (FDR—Benjamini–Hochberg (BH) adjustment) and fold-change > (1.3) were regarded as differentially expressed. Classification of differentially expressed genes (DEGs) and functional enrichment was performed with the DAVID functional annotation tool (https://david.ncifcrf.gov/summary.jsp ) and STRING version 11.0 (https://string-db.org/cgi/input.pl ? sessionId = OEsGZBwjRNJY). GSEA (gene set enrichment analysis) was performed using WebGestalt software (http://www.webgestalt.org/ ). To compare the RNA-seq results with the qPCR results, the Pearson correlation between the fold change obtained after RNA-seq and qPCR was calculated using SAS software. Statistical analysis of gene expression after the qPCR analysis was performed with the Mann–Whitney test.
10
Illumina HiSeq 2500 Rapid Sequencing
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Sequencing was performed using an Illumina HiSeq 2500 in Rapid mode employing a paired-end, 50 base read length (PE50) sequencing strategy. Image analysis and base calling was performed using Illumina’s Real Time Analysis v1.18 software, followed by ‘demultiplexing’ of indexed reads and generation of FASTQ files, using Illumina’s bcl2fastq Conversion Software v1.8.4.
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