Organoid RNA was isolated and treated with DNAse with Quick-RNA Micro prep kit (Zymo Research) according to manufacturer's instructions. RNA integrity numbers were found to be ≥7. For the I-CBP112 inhibitor study, library preparation was done using the Illumina TruSeq Stranded protocol. Library concentrations were quantified with the Qubit Fluorometric Quantitation system (Life Technologies) and the size distribution was assessed using a 2100 Bioanalyzer automated electrophoresis system (Agilent). For sequencing, samples were diluted and pooled into NGS libraries in equimolar amounts and sequenced at 75 bp single-read chemistry on an Illumina NS500 MO flow-cell on a Ilumina NextSeq 500 instrument (Illumina) by the Genomics core facility (GCF, NTNU). For the MS023 study, library preparation was done using the NEB Next Ultra RNA Library Prep Kit with poly(A) mRNA enrichment and samples were sequenced at 150X2 bp paired-end chemistry on a Illumina NovaSeq 6000 instrument by Novogene (UK) Co.
Truseq stranded protocol
Manufactured by Illumina
Sourced in United States
The TruSeq Stranded protocol is a laboratory tool designed for the preparation of stranded RNA-sequencing libraries. It provides a method for generating sequencing-ready libraries from total RNA or poly(A) enriched RNA samples. The core function of the TruSeq Stranded protocol is to preserve the original orientation of the RNA transcripts, enabling the identification of the strand from which each transcript was derived.
Automatically generated - may contain errors
Lab products found in correlation
Ns500 mo flow cell, by Illumina (3 mentions)
Quick rna microprep kit, by Zymo Research (3 mentions)
Hiseq 2500, by Illumina (2 mentions)
Ribo zero gold, by Illumina (2 mentions)
Nebnext ultra rna library prep kit, by New England Biolabs (2 mentions)
Qubit fluorometric quantitation system, by Thermo Fisher Scientific (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
Nextseq 500, by Illumina (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Trizol chloroform, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Nextseq 550 system, by Illumina (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Ribo zero kit, by Illumina (1 mentions)
2100 bioanalyzer instrument, by Agilent Technologies (1 mentions)
Hiseq 2500 platform, by Illumina (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Rneasy lipid tissue mini kit, by Qiagen (1 mentions)
10 protocols using truseq stranded protocol
1
Organoid RNA Sequencing Protocols
2
Transcriptome Profiling via RNA-Seq
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Total RNAs were isolated using the RNeasy Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions in conjunction with on-column DNase digestion to remove genomic DNA contamination. RNA from two biological replicates for each condition was analyzed. Total RNA samples were then sent to the Yale Center for Genome Analysis for whole transcriptome sequencing. Strand-specific polyA-RNA libraries were prepared by the Illumina TruSeq stranded protocol. The libraries underwent 75 bp paired-end reads using an Illumina HiSeq 2500 according to Illumina protocols, generating an average of 35 million paired-end reads per library.
3
Comparative Camel Genome Assembly Evaluation
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To assess the quality of the new assembly, we aligned 10 sets of paired‐end RNA‐Seq reads (Alim et al., 2019 ) to the original assembly (CamDro1), to the new assembly (CamDro2), and to several controls: C. dromedarius (RefSeq version ‐ GCA_000767585.1 ), C. bactrianus (GCA_000767855.1 ), C. ferus (GCA_000311805.2 ) and Bos taurus (cattle) (GCA_000003055.3 ). The 10 RNA‐Seq data sets comprise a 2 × 2 factorial experiment: summer versus winter seasons and supraoptic nucleus (SON) versus neurointermediate lobe (NIL) brain tissues, with n = 3 replicates in each class. Tissue was homogenized and extracted using in Trizol/chloroform (ThermoFisher), and purified using the RNeasy MiniKit (Qiagen). The library template was prepared using a ribosome depletion protocol (Ribo‐Zero Gold; Illumina) and libraries prepared using TruSeq Stranded protocol (Illumina). Samples were multiplexed into lane pools with an 8 pm concentration and sequenced (100 bp paired‐end reads with an average 134 bp insert size) to a depth of > 35 million reads using an Illumina HiSeq 2500. Two of the 12 replicates were rejected for insufficient quality. We used tophat v. 2.0.9 (Kim et al., 2013 ) with default settings to align reads to each genome and report overall alignment rate (default output of Tophat) within each class.
4
RNA-seq Library Preparation and Sequencing
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The IBENS Genomics Facility conducted the RNA-seq library construction. The integrity of isolated total cytoplasmic RNA was verified using the RNA Pico method on the Agilent 2100 Bioanalyzer. High-quality RNA (RIN > 8) was used in library preparation with the Illumina TruSeq stranded protocol (Illumina, San Diego, USA). Libraries were rRNA depleted using the Illumina Ribo Zero kit and sequenced as single read 75 base pair read length (SR75) on the NextSeq 550 system by the IBENS Genomics Facility.
5
Oxaliplatin-Induced DRG Transcriptome Analysis
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To investigate global mRNA changes, we used RNA sequencing analysis on L4-L5-L6 DRGs from mice treated with oxaliplatin (3 mg kg−1, i.p.) or vehicle twice per week for 3 weeks. At the end of the experiment (D21), mice were terminally anesthetized and L4-L5-L6 DRGs were quickly harvested, snap frozen in liquid nitrogen, and stored at −80 °C until use. Total RNA of L4-L5-L6 DRGs was extracted using RNeasy Micro Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Thereafter, rRNA was removed using Illumina® Ribo-Zero Plus rRNA Depletion Kit (Epicentre, Illumina, San Diego, CA, USA). RNA integrity and concentration were obtained using 2100 Bioanalyzer instrument (Agilent Technologies, Santa Clara, CA, USA) and were sent to Fasteris (https://www.fasteris.com (accessed on 6 September 2017)) for RNA sequencing. Libraries were prepared using the Illumina TruSeq stranded protocol, and an SBS-based sequencing was achieved using HiSeq 2500 platform (Illumina). Analysis was performed in different steps: splice junction mapping (TopHat2), counting (HTSeq-count), filtering, normalization (edgeR, DESeq and DESeq2) and differential analysis (edgeR, DESeq and DESeq2) were performed by Benjamin Bertin and Yoan Renaud (GreD, Clermont-Ferrand, France).
6
RNA-seq Analysis of Transcriptome
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RNA was isolated using RNeasy Plus Mini Kit (Qiagen; catalog no. 74034) according to the manufacturer's instructions. Total RNA was sequenced at the MD Anderson ATGC Sequencing Core Facility with NovaSeq, producing 100-bp pair end reads. The sample library was prepared using Illumina TruSeq stranded protocol. RNA-seq FASTQ files were processed through FastQC to assess the quality of sequencing reads. Samples passed QC were used for analysis. STAR alignment to the GRCm38 was achieved with default parameters to generate RNA-seq BAM files. Aligned reads were summarized at the gene level using STAR. Gene-level annotation was performed using the GENCODE annotation, which was downloaded from the GENCODE project. The final P-value was adjusted using the Benjamini and Hochberg method.
7
Transcriptome Profiling of Tissue Samples
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The total RNA was extracted with HiPure Universa miRNA Kit (Magen) from two pairs of fresh frozen tissue samples and quality was confirmed by Nanodrop measurement of OD 260/280 and 260/230 ratios. The material for library construction was 1 μg per sample. Sequencing libraries were constructed following the Illumina TruSeq Stranded protocol. Total RNA Gold kit with Ribo-Zero Gold (Illumina, USA) was used following the manufacturer’s recommendations. Sequencing (2×150 paired-end reads) was performed at Mingma Technologies Co., Ltd in Shanghai.
FASTQ format data were assessed using FastQC (v0.11.9) and then fastp (v0.20.1) (35 (link)) was used to remove the bases with an average quality value less than 20 and to cut the reads of adapters. Clean reads were mapped to the human reference genome (Gencode v19) by STAR (2.7.5b) (36 (link)). The gene and transcript isoform expression was quantified using RSEM (v1.3.1) (37 (link)). Bedtools (v2.29.2) (38 (link)) was used to transform the bam files to bw format for UCSC genome browser viewing.
FASTQ format data were assessed using FastQC (v0.11.9) and then fastp (v0.20.1) (35 (link)) was used to remove the bases with an average quality value less than 20 and to cut the reads of adapters. Clean reads were mapped to the human reference genome (Gencode v19) by STAR (2.7.5b) (36 (link)). The gene and transcript isoform expression was quantified using RSEM (v1.3.1) (37 (link)). Bedtools (v2.29.2) (38 (link)) was used to transform the bam files to bw format for UCSC genome browser viewing.
8
RNA Isolation and Sequencing of Intestinal Epithelial Cells
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RNA for WT and LSD1KO crypts was isolated by sorting IECs (DAPI−, Epcam+) in 2× RNA shield buffer (Zymo) and RNA isolation using the Quick-RNA Microprep Kit (Zymo). Library preparation was done using the Illumina TruSeq Stranded protocol, and samples were sequenced at 75 × 2–base pair (bp) paired-end (PE) reads on an Illumina NS500 MO flow cell. Sequencing was performed by the Genomics Core Facility (NTNU). IECs from E18.5, P7, and E18.5 were directly dissolved in RNA isolation buffer, and RNA was isolated using the Quick-RNA Microprep Kit (Zymo). Library preparation was done using the NEBNext Ultra RNA Library Prep Kit. Sequencing was performed by Novogene (UK) Co.
9
Transcriptomic Analysis of Fam13a Knockout in Adipose Tissue
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RNA was extracted from ~50 mg of liver, visceral and subcutaneous adipose tissues using RNeasy Lipid Tissue Mini Kit (Qiagen). Library preparation and RNA sequencing were carried out by using the Illumina TruSeq Stranded protocol, with an average number of reads per sample as 40 million. Reads were aligned to the Grcm38 mouse genome using STAR 2.6 software48 (link), and Gencode M11 mouse gene annotations and counts were obtained using the quantMode option in STAR. Differential expression analysis was performed using edgeR 3.20.949 (link) with a false discovery rate (FDR) of 10%. Analysis of the transcriptome-wide effects of Fam13a KO was performed in adipose tissue samples from both SAT and VAT fat depots of male mice, adjusted for known (diet, fat depot, RIN, number of days from tissue harvest to RNA extraction) and hidden covariates (6 PEER factors).
10
RNA-Seq analysis of LSD1 knockout crypts
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RNA for WT and LSD1KO crypts was isolated by sorting IECs (DAPI-, Epcam+) in 2x RNA shield buffer (Zymo) and RNA isolation using the Quick-RNA Micro prep kit (Zymo).
Library preparation was done using the Illumina TruSeq Stranded protocol, and samples were sequenced at 75X2 bp PE reads on an Illumina NS500 MO flow-cell. Sequencing was performed by the Genomics core facility (GCF, NTNU). Crypts from E18.5, P7 and E18.5 was directly dissolved in RNA isolation buffer and RNA was isolated using the Quick-RNA Micro prep kit (Zymo). Library preparation was done using the NEB Next® Ultra™ RNA Library Prep Kit. Sequencing was performed by Novogene (UK) Co.
Library preparation was done using the Illumina TruSeq Stranded protocol, and samples were sequenced at 75X2 bp PE reads on an Illumina NS500 MO flow-cell. Sequencing was performed by the Genomics core facility (GCF, NTNU). Crypts from E18.5, P7 and E18.5 was directly dissolved in RNA isolation buffer and RNA was isolated using the Quick-RNA Micro prep kit (Zymo). Library preparation was done using the NEB Next® Ultra™ RNA Library Prep Kit. Sequencing was performed by Novogene (UK) Co.
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