The enriched total nucleic acid (11 μL) from each tick pool was then subjected to first- and second-strand cDNA synthesis with Super Script IV reverse transcriptase (Invitrogen, Waltham, MA, USA) and exo-Klenow fragment (New England Biolabs, Ipswich, MA, USA), respectively. For all libraries, the Ribo-Zero Gold Kit (Illumina) was used to remove ribosomal RNA under the manufacturer's guidance. Subsequently, all rRNA-depleted RNA samples were resuspended to construct libraries using the KAPA Stranded RNA-Seq Kit (KAPA biosystems, Roche) with TruSeq Index PCR Primer barcodes (Illumina, SanDiego, CA, USA) following the manufacturer's instructions. Qubit high-sensitive RNA assays (Thermo Fisher Scientific) were performed to quantify cDNA levels before, during, and after library preparation, and the fragment sizes were simultaneously determined with an Agilent Bioanalyzer. Subsequently, equimolar amounts of nucleic acids were pooled and submitted for sequencing in each library. All libraries were sequenced on a single lane (paired-end, 125 bp read-length) on an Illumina HiSeq 2,500 platform at the BGI Sequencing Center (www.genomics.cn ).
Kapa stranded rna seq kit
Manufactured by Roche
Sourced in United States, Switzerland
The KAPA Stranded RNA-Seq Kit is a library preparation kit designed for generating stranded RNA-sequencing libraries. The kit includes reagents and protocols for converting total RNA into a sequencing-ready library, with the ability to preserve the original orientation of the RNA transcripts.
Automatically generated - may contain errors
Lab products found in correlation
Agilent 2100 bioanalyzer, by Agilent Technologies (10 mentions)
Rneasy mini kit, by Qiagen (9 mentions)
Hiseq 2500, by Illumina (8 mentions)
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Hiseq 4000, by Illumina (4 mentions)
Agencourt rnaclean xp beads, by Beckman Coulter (3 mentions)
Nextseq, by Illumina (3 mentions)
2100 bioanalyzer, by Agilent Technologies (3 mentions)
Novaseq 6000 system, by Illumina (3 mentions)
Riboerase, by Roche (3 mentions)
Miseq reagent kit v2, by Illumina (3 mentions)
Nextseq 500, by Illumina (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Hybridase thermostable rnase h, by Illumina (2 mentions)
Rnaclean xp beads, by Thermo Fisher Scientific (2 mentions)
Kapa hyperprep rna seq kit, by Roche (2 mentions)
Turbo dnase, by Thermo Fisher Scientific (2 mentions)
Nimblegen seqcap ez medexome target enrichment probes, by Roche (2 mentions)
Miseq instrument, by Illumina (2 mentions)
X ten pe150 platform, by Illumina (2 mentions)
52 protocols using kapa stranded rna seq kit
1
Tick transcriptome profiling by RNA-seq
2
Gene Expression and Cytokine Profiling
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To ensure data quality, the changes in expression of representative inflammatory genes were confirmed using RT-qPCR (Supplementary Table S1 ). The primer sequences used in the RT-qPCR experiments are listed in Supplementary Table S2 . The production of inflammatory cytokine IL-1β and chemokine IL-8 from the skin culture medium were measured using ELISA (Supplementary Table S3 ).
RNA-Seq library construction was performed as described previously [19 (link)] using 1–2 µg of each RNA sample. Poly-A tailed RNA enrichment was done using the Magnetic mRNA Isolation Kit (New England Biolabs, Ipswich, MA, USA). To prepare complementary DNA libraries, mRNAs were enzymatically fragmented followed by first and second strand complementary DNA synthesis and unique indices were ligated using the Kapa Stranded RNA-Seq Kit (Kapa Biosystems, Wilmington, MA, USA). DNA libraries were amplified by polymerase chain reaction followed by cleaning and size selection using the AMPure XP kit (Agencourt Bioscience, Beverly, MA, USA). DNA samples were quantified using the Quant-iT™ dsDNA Assay Kit (ThermoFisher Scientific) and normalized to 4 nM. RNA-Seq libraries were sequenced on a HiSeqX sequencer (Illumina) at Canada’s Michael Smith Genome Sciences Centre at the British Columbia Cancer Agency.
RNA-Seq library construction was performed as described previously [19 (link)] using 1–2 µg of each RNA sample. Poly-A tailed RNA enrichment was done using the Magnetic mRNA Isolation Kit (New England Biolabs, Ipswich, MA, USA). To prepare complementary DNA libraries, mRNAs were enzymatically fragmented followed by first and second strand complementary DNA synthesis and unique indices were ligated using the Kapa Stranded RNA-Seq Kit (Kapa Biosystems, Wilmington, MA, USA). DNA libraries were amplified by polymerase chain reaction followed by cleaning and size selection using the AMPure XP kit (Agencourt Bioscience, Beverly, MA, USA). DNA samples were quantified using the Quant-iT™ dsDNA Assay Kit (ThermoFisher Scientific) and normalized to 4 nM. RNA-Seq libraries were sequenced on a HiSeqX sequencer (Illumina) at Canada’s Michael Smith Genome Sciences Centre at the British Columbia Cancer Agency.
3
Chlamydomonas Sexual Cycle Transcriptome
4
Total RNA Sequencing for Transcriptome Analysis
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For total RNA sequencing (RNA-seq), RNA was isolated as described in Text S1 . Illumina system-compatible sequencing libraries were prepared essentially as described previously (50 (link)). RNA quantity and integrity were assessed using an Agilent 2100 Bioanalyzer (Agilent RNA 6000 Nano kit). Briefly, RNA samples were initially purified using magnetic beads (AMPure XP magnetic beads; Becton, Dickinson), rRNA was removed (Ribo-Zero rRNA removal kit; Illumina), and sequencing libraries were generated (KAPA stranded RNA-Seq kit; KAPA Biosystems) according to the manufacturer’s protocol. Illumina system-compatible adapters containing sample-specific 8-nucleotide-long barcoding sequences were ligated, and the cDNA libraries were PCR amplified. The resulting sequencing libraries were evaluated using an Agilent 2100 Bioanalyzer with a DNA 1000 chip and quantified by real-time PCR using the NEBNext Library Quant kit for Illumina (New England Biolabs). Raw sequencing data were generated on a NextSeq500 platform (Illumina) using paired-end 75-cycle sequencing run-compatible reagents (150 cycles, NextSeq 500/550 Mid Output v2 sequencing kit; Illumina). Libraries were prepared and sequenced in biological triplicates for each tested growth condition.
5
Drosophila rRNA Depletion for RNA-seq
6
RNA Extraction and Library Preparation for Hepatic Transcriptome Sequencing
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Total RNA was extracted from hepatic tissue using an RNAeasy extraction kit from QIAGEN®30 with a DNAse cleaning step according to the manufacturer’s instructions. RNA quality was measured using Nanodrop, Qubit 2.0 fluorometer and Bioanalyzer 2100 instruments.
Fifteen libraries (one per individual) were constructed from 1.0 µl of total RNA using the KAPA Stranded RNA-Seq kit with RiboErase from KapaBiosystems®. Ribosomal RNA was removed by depletion by DNA primer hybridization followed by treatment with RNAse and DNAse according to the manufacturer’s instructions. The fragmentation cycle was adjusted to 10 cycles of amplification at 94 °C for 5 minutes to obtain fragments between 100 and 2100 bp in length. Each library was enriched with Illumina® TruSeq Index Adapters (250 nM) for multiplex sequencing.
Library quality was evaluated using a Qubit 2.0 fluorometer and an Agilent Bioanalyzer 2100; good quality libraries were paired-end (PE) sequenced on an Illumina HiSeq. 4000 at the Center for Genomics Services of the University of California, Berkeley.
Fifteen libraries (one per individual) were constructed from 1.0 µl of total RNA using the KAPA Stranded RNA-Seq kit with RiboErase from KapaBiosystems®. Ribosomal RNA was removed by depletion by DNA primer hybridization followed by treatment with RNAse and DNAse according to the manufacturer’s instructions. The fragmentation cycle was adjusted to 10 cycles of amplification at 94 °C for 5 minutes to obtain fragments between 100 and 2100 bp in length. Each library was enriched with Illumina® TruSeq Index Adapters (250 nM) for multiplex sequencing.
Library quality was evaluated using a Qubit 2.0 fluorometer and an Agilent Bioanalyzer 2100; good quality libraries were paired-end (PE) sequenced on an Illumina HiSeq. 4000 at the Center for Genomics Services of the University of California, Berkeley.
7
Barcode Indexed RNA-seq Library Construction
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The construction of barcode indexed RNA-seq libraries and deep sequencing were performed by UC Davis genome center DNA core facility. In brief, poly-A RNA was enriched from 1 μg of total RNA with Kapa Stranded RNA-seq kit (KapaBiosystems, Cape Town, South Africa). The RNA-seq libraries were generated on a Sciclone NGS G3 liquid handler (Caliper Life Sciences, Alameda, CA, USA) and constructed libraries were analyzed with a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA) followed by the fluorometric quantification by Qubit (LifeTechnologies, Carlsbad, CA, USA). Libraries were pooled in equimolar ratios, quantified by qPCR with a Kapa Library Quant kit (KapaBiosystems), and sequenced on Illumina HiSeq 3000 platform (Illumina, San Diego, CA, USA) with paired-end 100 bp reads.
8
Transcriptome RNA Sequencing for MAPK Alterations
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For the nine cases in which a pathogenic MAPK activating alteration was not identified by DNA sequencing, whole transcriptome RNA sequencing was performed. 170–400 ng of total RNA was prepared for sequencing using the KAPA stranded RNA-seq kit (KAPA Biosciences, Wilmington, MA, p/n KK8400) according to the manufacturer’s instructions. Subsequently, exome capture was carried out using NimbleGen SeqCap EZ MedExome target enrichment probes (Roche, Basel, Switzerland, p/n 07681330001) following the manufacturer’s protocol. Sequencing was performed on a NovaSeq 6000 system (Illumina Inc, San Diego, CA) with 150-bp paired-end reads. Sequence reads were mapped to the reference human genome (hg19) using STAR44 (link) and fusions were identified by FusionCatcher45 and verified by manual review.
9
Paired-end Illumina RNA-Seq Library Preparation
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Paired-end sequencing was conducted from cDNA libraries prepared from total RNA using KAPA Stranded RNA-Seq kit (KAPA Biosystems, USA) as per manufacturer’s instructions. Briefly, the protocol involved the synthesis of both the first strand and second strand of DNA from total extracted RNA by using random-priming, marking and A-tailing of cDNA fragments with 3′-adenine residues to allow ligation of 3′-thymine-containing adaptors, and random PCR amplification to create the DNA library. The fragment length distribution for each library was assessed using the Agilent High Sensitivity DNA Kit (Agilent Technologies, USA) on the Agilent 2100 Bioanalyzer (Agilent Technologies, USA). The Qubit® fluorometer and the Qubit® dsDNA HS Assay Kit was used to measure the concentration of the dsDNA libraries. All libraries for NGS underwent equimolar dilution and were pooled. The library pool was loaded into the 300 cycle MiSeq Reagent Kit v2 (Illumina, USA) and pair-end sequencing (2 × 150 bp) was performed on the Illumina MiSeq instrument (Illumina, USA). After automated cluster generation in MiSeq, the sequencing reads were processed by bioinformatics. Pre-processing and de-novo assembly of the raw sequencing data was completed within the Galaxy platform [20 (link)] using a previously described approach [17 (link)].
10
Comparative NGS Analysis of NDV Isolates
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For comparison between nucleotide sequences obtained from MinION and MiSeq (a high accuracy sequencing platform), 24 NDV isolates from the SEPRL repository that were used for MinION sequencing (representing each currently circulating genotype except XI and multiple sub-genotypes of NDV) and 15 clinical swab samples (allantoic fluid of cultured swab samples) were processed for target-independent NGS sequencing. Briefly, paired-end random sequencing was conducted from cDNA libraries prepared from total RNA using KAPA Stranded RNA-Seq kit (KAPA Biosystems, USA) as per manufacturer’s instructions and as previously described [46 (link)]. All libraries for NGS were loaded into the 300-cycle MiSeq Reagent Kit v2 (Illumina, USA) and pair-end sequencing (2 × 150 bp) was performed on the Illumina MiSeq instrument (Illumina, USA). Pre-processing and de-novo assembly of the raw sequencing data was completed within the Galaxy platform using a previously described approach [19 (link)].
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