Ribosomal RNA-depleted cDNA libraries were prepared using KAPA RNA HyperPrep kits with RiboErase (KAPA Biosystems) according to the manufacturer’s protocol. Libraries were sequenced with an Illumina Hi-seq 3000 (single-end, 1 × 100 bp).
Kapa rna hyperprep kit
Manufactured by Roche
Sourced in United States, Switzerland, Germany
The KAPA RNA HyperPrep kit is a RNA library preparation kit designed for high-throughput sequencing applications. The kit enables efficient conversion of RNA samples into DNA libraries suitable for sequencing on Illumina platforms.
Automatically generated - may contain errors
Lab products found in correlation
Novaseq 6000, by Illumina (16 mentions)
Trizol, by Thermo Fisher Scientific (12 mentions)
Rneasy mini kit, by Qiagen (11 mentions)
Nextseq 500, by Illumina (10 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (9 mentions)
Rneasy plus mini kit, by Qiagen (8 mentions)
Riboerase, by Roche (7 mentions)
Hiseq platform, by Illumina (7 mentions)
Kapa dual indexed adapter kit, by Roche (6 mentions)
Novaseq platform, by Illumina (6 mentions)
Trizol reagent, by Thermo Fisher Scientific (6 mentions)
Quantifluor dsdna system, by Promega (5 mentions)
Qiaseq fastselect rrna hmr kit, by Qiagen (5 mentions)
Agilent dna high sensitivity chip, by Agilent Technologies (5 mentions)
Truseq ud indexed adapters, by Illumina (5 mentions)
Hiseq 4000, by Illumina (5 mentions)
Rnaprotect bacteria reagent, by Qiagen (5 mentions)
Agilent bioanalyzer dna 1000 chip, by Agilent Technologies (5 mentions)
Kapa library quantification kit, by Roche (5 mentions)
Ribo zero gold rrna removal kit, by Illumina (5 mentions)
141 protocols using kapa rna hyperprep kit
1
Ribosomal RNA Depletion for Transcriptome Analysis
2
RNA Extraction and Library Preparation
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Sorted populations were resuspended in Trizol LS (ThermoFisher) and RNA isolated according to the manufacturer’s instructions. 3′ end capture libraries were prepared using Quant-seq (Lexogen) and full-length libraries using Kapa RNA Hyper-prep kits (Kapa Biosystems), per manufacturer’s instructions. Single-end 50 bp sequencing of these libraries was then performed on a HiSeq4000.
3
Ribosomal RNA Depletion for Transcriptome Analysis
4
Aortic RNA-seq Analysis Pipeline
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Fresh-frozen aortic samples for RNA-seq were obtained from independent cohorts of patients with similar clinical and demographic characteristics (Supplementary Table 1). Total RNA was extracted and evaluated for quality as described above. RNA-seq library preparation was performed using KAPA RNA HyperPrep Kits. All libraries were pooled and sequenced on 1 lane of a HiSeq 4000 instrument. The samples were sequenced to a mean depth of 30.86 million reads. FASTQC was utilized to inspect the raw sequencing data. Spliced Transcripts Alignment to a Reference (STAR, ver2.6) [19] (link) was used to align the sequence reads to hg19, and RSEM (ver1.3.0) [20] (link) was used to estimate the gene level abundance. Gene level count data were processed and normalized, and between-group analyses were performed in DESeq2 [21] (link). Weakly expressed genes with fewer than 80 counts in 50% of study samples were excluded from analysis. The remaining 8610 genes were subsequently utilized to identify DEGs between HSS and LSS groups. For validation of the genes identified from the discovery microarray analysis, the direction of effect was required to be consistent between the SS study groups, and the association levels were suitably adjusted for the number of multiple tests performed using a Bonferroni p value correction (Supplementary Fig. 2).
5
Transcriptome Profiling of Cells and Brain
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RNA from cells and brain was prepared as RNA-seq libraries using Kapa RNA HyperPrep kits (Roche, Basel, Switzerland) together with the QIAseq FastSelect Human ribodepletion kit (Qiagen, Hilden, Germany). Libraries were assessed for quality and quantified on the Agilent Bioanalyzer 2100, and then pooled for multiplex sequencing with at least 25 million reads with 150 bp paired-end reads on the Illumina NovaSeq 6000 S4 (San Diego, CA, USA) by the DNA Tech Core at University of California, Davis (Davis, CA, USA).
6
Rapid TT-seq Library Preparation
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TT-seq libraries were prepared following a published protocol31 (link),. The untreated and 500 nM treated WAPL-AID cells (6 and 24 h) were labeled using 2 mM 4SU for 10 min. Total RNA from the labeled cells was isolated and fragmented. Then 4SU-labeled RNA fragments were biotinylated and enriched using streptavidin MicroBeads. TT-seq libraries were prepared using KAPA RNA HyperPrep Kits (Roche) and KAPA Dual-Indexed Adapter Kits (Roche). TT-seq libraries were sequenced on a NextSeq 550.
7
Rapid TT-seq Library Preparation
8
Temporal Transcriptome Analysis of SOX2‐FKBP Cells
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Libraries for TTchemseq were prepared following a published protocol (Gregersen et al, 2020 (link)). SOX2‐FKBP cells were seeded and the day after treated with 500 nM of dTAG‐13 (0.5, 1, 2, 6 hpd) or DMSO as control. Cells were labeled with 2 mM 4SU for 10 min. Total RNA was isolated and fragmented. The 4SU‐biotin labeled RNA was enriched using streptavidin coated MicroBeads. Libraries were prepared using KAPA RNA HyperPrep kits (Roche) using dual indexing adapters. Libraries were sequenced on a NextSeq 550.
9
RNA-Seq Library Preparation and Data Analysis
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RNA was extracted by RNeasy mini plus kit (QIAGEN). mRNA selection was performed using NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs). Library preparation for RNA-Seq analysis was conducted with the KAPA RNA HyperPrep Kit (KAPA BIOSYSTEMS). RNA-seq libraries were sequenced on the Illumina NextSeq 550. Sequence reads were aligned to the GENCODE human reference genome GRCh38 using STAR55 (link) (v. 2.6.1d). Aligned reads were counted by RSEM56 (link) (v1.3.1). The count data normalized using the DESeq2 package57 (link) (v1.32.0) were subjected to GSEA31 (link). cDNA was synthesized using iScript reverse transcription supermix for RT-qPCR (Bio-Rad Laboratories). qRT-PCR was performed with iQ SYBR green supermix kit and the CFX96 Real-time PCR system (Bio-Rad Laboratories). All primers used in this paper are listed in the Supplemental Table S6 .
10
Bulk RNA-seq Library Preparation and Analysis
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For bulk RNA-seq, total RNA concentration was calculated by Quant-IT RiboGreen (Invitrogen, #R11490). To assess the integrity of the total RNA, samples were run on the TapeStation RNA screentape (Agilent, #5067-5576). Only high-quality RNA preparations, with RIN greater than 7.0, were used for RNA library construction. Ribosomal RNA was removed before cDNA synthesis with SuperScript II reverse transcriptase (Invitrogen) and library preparation with KAPA RNA Hyper Prep Kit (Kapa Biosystems). RNA libraries were prepared according to the manufacturers’ protocol (Kapa Biosystems). Libraries were pooled together for pair-end sequencing with NovaSeq 6000 or NextSeq 500 (High output) platform (illumina).
Pair-end sequencing reads of RNA-seq data were mapped to human genome (hg19 version) or mouse genome (mm10 version, for mouse bulk RNA-seq) with tophat software (tophat2, default parameter)81 (link), annotated repeat information was downloaded from UCSC database, repeat region quantification was done with BEDTOOLs82 (link) and normalized to cpm (count per million for each sample). Gene expression was calculated with cufflinks software83 (link). Kolmogorov–Smirnov test (KS test) was used for comparing transposon fold-change to the background. Differential expression analysis was done using DESeq284 (link).
Pair-end sequencing reads of RNA-seq data were mapped to human genome (hg19 version) or mouse genome (mm10 version, for mouse bulk RNA-seq) with tophat software (tophat2, default parameter)81 (link), annotated repeat information was downloaded from UCSC database, repeat region quantification was done with BEDTOOLs82 (link) and normalized to cpm (count per million for each sample). Gene expression was calculated with cufflinks software83 (link). Kolmogorov–Smirnov test (KS test) was used for comparing transposon fold-change to the background. Differential expression analysis was done using DESeq284 (link).
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