Sequel ® Sequencing Kit 2.1 (PacBio) was used to construct an SMRT library, and high-quality long reads were generated using an RSII system. For Hi-C, libraries were performed according to a previously described method (Wang et al., 2020 (link); Wu et al., 2020 (link)), via HindIII digestion and sequencing using a Hiseq X platform (Illumina) with an average depth of 69 ×. Poly (A) strand-specific libraries were constructed using the KAPA Stranded mRNA-Seq Library Preparation kit (KK8421; Roche, Pleasanton, CA, United States). All libraries were sequenced using an Illumina Hiseq 4000 sequencing system.
Kapa stranded mrna seq library preparation kit
Manufactured by Roche
Sourced in United States, Switzerland
The KAPA Stranded mRNA-Seq Library Preparation kit is a laboratory equipment product designed for the preparation of stranded mRNA sequencing libraries. The kit provides the necessary reagents and protocols for the isolation, fragmentation, and conversion of mRNA into sequencing-ready libraries.
Automatically generated - may contain errors
Lab products found in correlation
Sequel sequencing kit 2, by Pacific Biosciences (1 mentions)
Hiseq 4000 sequencing system, by Illumina (1 mentions)
Hiseq x platform, by Illumina (1 mentions)
Hiseq platform, by Illumina (1 mentions)
Specific taqman probes, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
Hiseq x pe cluster kit v2, by Illumina (1 mentions)
Cbot cluster generation system, by Illumina (1 mentions)
Trizol, by Transgene (1 mentions)
Hiseq 2500 platform, by Illumina (1 mentions)
4 protocols using kapa stranded mrna seq library preparation kit
1
Long-read Sequencing and Hi-C Profiling
2
Transcriptome assembly and annotation
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The transcriptome was assembled using the six libraries prepared with the Kapa stranded mRNAseq Library Preparation kit (Roche) to minimize the sequencing bias for high GC content regions. Libraries were sequenced using the Illumina HiSeq platform at the GTF resulting in total 192 million reads of size 2 × 100 bp summing up to 34 Gbp per tissue (Table S4 ). The reads were assembled using Trinity (Haas et al., 2013 ) (version 2014.07.17) with default parameters and the option trimmomatic for quality trimming reads before assembling them. The resulting transcriptome consisted of 421,658 contigs ranging from 201 to 21,648 bp with a mean of 808 bp and a median of 382 bp. The transcriptome (Tyto_Alba_DEE_transcriptome.fasta) was filtered for transcripts with homologies to known proteins, by first extracting long open reading frames (ORFs) followed by a blast and a pfam search to known proteins (Haas et al., 2013 ).
3
Transcriptome Analysis of Lung Tissue
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RNA was extracted from lung tissue using Trizol (Invitrogen) and the RNeasy kit (Qiagen). Quantitative RT-PCR was performed using the Omniscript reverse transcription kit, and amplification was performed using the Veriquest PCR master mix and specific Taqman probes from Applied Biosystems. Expression relative to the human RPL7 or mouse Rpl7 ‘housekeeping’ gene was calculated using the Delta Delta Ct method. RNA sequencing was performed using the KAPA Stranded mRNA-Seq Library Preparation kit (Kapa Biosystems) following the manufacturer’s instructions using an Illumina HiSeq 2000 or HiSeq 2500 platform (Illumina, San Diego, CA). Sequenced reads (50 bp, single end) were mapped to the mouse genome (NCBI37/mm9, July 2007) using Bowtie 0.12.9, and only the reads that mapped onto exons of each RefSeq gene were measured and normalized using reads per kilobase per million mapped reads. Analyses of differential gene expression were performed using R package edgeR.
4
Total RNA Extraction and RNA-seq Library Preparation
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Total RNA was extracted as previously described (Peng et al., 2021 (link)). Briefly, approximately 0.1 g of symptomatic leaves per sample was ground into a homogenate in liquid nitrogen, and 1 mL of TRIzol (TransGen, Beijing, China) was added to each sample. The total RNA–containing supernatant was washed with an equivalent volume of chloroform; the total RNA was precipitated using isopropanol and dissolved in RNase-free water with RNase inhibitors. The total RNA quality and quantity were assessed using a SpectraMax NanoDrop system (Thermo Fisher Scientific, MA, USA). All samples had RNA integrity numbers higher than 8.0, indicating relatively intact and protein-free RNA. The RNA-seq library was prepared using 4 μg total RNA, with the KAPA Stranded mRNA-Seq Library Preparation Kit (Kapa Biosystems, Roche, Basel, Switzerland). RNA-seq libraries were used for cluster formation with the HiSeq X PE Cluster Kit V2.5 on an Illumina cBOT cluster generation system (Illumina) and sequenced in one lane by using the Illumina HiSeq 2500 platform in the 100 bp paired-end mode.
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