RNA quality was checked using RNA Integrity Number (RIN) from Bioanalyser and 500 ng of RNA was used to prepare libraries using Illumina TruSeq stranded mRNA (HT) library preparation kit. Library size distribution was assessed using the Agilent Tapestation 4200 system. These were sequenced using HiSeq 4000 50 bp single end sequencing. 1% PhiX version 3 viral genome spike-in was introduced during sequencing. Fastaq single-end reads from multiple lanes were merged to make a single library per replicate. STAR40 (link), version 2.5.1a, was used to align reads against hg38 reference genome. The read counting was performed using the intrinsic function of STAR. Differential gene expression analysis used the DESeq2 workflow. All p-values were corrected for multiplicity by means of the Benjamini and Hochberg FDR multiplicity correction.
Truseq stranded mrna ht library preparation kit
Manufactured by Illumina
The TruSeq Stranded mRNA (HT) library preparation kit is a reagent system designed for the preparation of stranded mRNA libraries for next-generation sequencing. The kit enables the construction of high-quality mRNA libraries from total RNA samples, preserving the strand orientation of the transcripts.
Automatically generated - may contain errors
Lab products found in correlation
Ht dna hisens reagent kit, by PerkinElmer (3 mentions)
D1000 screentape and reagents, by Agilent Technologies (3 mentions)
Hiseq 3000 4000 sbs kit, by Illumina (3 mentions)
Hiseq 3000 4000 pe cluster kit, by Illumina (3 mentions)
Hiseq 4000 sequencer, by Illumina (3 mentions)
Labchip gx bioanalyser, by PerkinElmer (3 mentions)
4200 tapestation system, by Agilent Technologies (2 mentions)
5 protocols using truseq stranded mrna ht library preparation kit
1
RNA-seq analysis of gene expression
2
cDNA Library Preparation from RNA
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cDNA library preparation: for blood and tissues, total/globin-reduced RNA (200 ng) was used to prepare cDNA libraries using the TruSeq Stranded mRNA HT Library Preparation Kit (Illumina). For cDNA library preparation of FACS sorted cells, total RNA (30–500 pg) was used to prepare cDNA libraries using the NEBNext® Single Cell/Low Input RNA Library Prep Kit NEBNext® Multiplex Oligos for Illumina® #E6609 (New England BioLabs). Quality and integrity of the tagged libraries were initially assessed with the HT DNA HiSens Reagent kit (Perkin Elmer) using a LabChip GX bioanalyser (Caliper Life Sciences/Perkin Elmer). Tagged libraries were then sized and quantitated in duplicate (Agilent TapeStation system) using D1000 ScreenTape and reagents (Agilent). Libraries were normalized, pooled and then clustered using the HiSeq® 3000/4000 PE Cluster Kit (Illumina). The libraries were imaged and sequenced on an Illumina HiSeq 4000 sequencer using the HiSeq® 3000/4000 SBS kit (Illumina) at a minimum of 25 million paired-end reads (75 bp/100 bp) per sample.
3
RNA-seq analysis of gene expression
4
cDNA Library Preparation for Single-Cell RNA-Seq
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cDNA library preparation: for blood and tissues, total/globin-reduced
RNA (200 ng) was used to prepare cDNA libraries using the TruSeq Stranded mRNA
HT Library Preparation Kit (Illumina). For cDNA library preparation of FACS
sorted cells, total RNA (30–500 pg) was used to prepare cDNA libraries
using the NEBNext® Single Cell/Low Input RNA Library Prep Kit
NEBNext® Multiplex Oligos for Illumina® #E6609 (New England
BioLabs). Quality and integrity of the tagged libraries were initially assessed
with the HT DNA HiSens Reagent kit (Perkin Elmer) using a LabChip GX bioanalyser
(Caliper Life Sciences/Perkin Elmer). Tagged libraries were then sized and
quantitated in duplicate (Agilent TapeStation system) using D1000 ScreenTape and
reagents (Agilent). Libraries were normalized, pooled and then clustered using
the HiSeq® 3000/4000 PE Cluster Kit (Illumina). The libraries were imaged
and sequenced on an Illumina HiSeq 4000 sequencer using the HiSeq®
3000/4000 SBS kit (Illumina) at a minimum of 25 million paired-end reads (75
bp/100 bp) per sample.
RNA (200 ng) was used to prepare cDNA libraries using the TruSeq Stranded mRNA
HT Library Preparation Kit (Illumina). For cDNA library preparation of FACS
sorted cells, total RNA (30–500 pg) was used to prepare cDNA libraries
using the NEBNext® Single Cell/Low Input RNA Library Prep Kit
NEBNext® Multiplex Oligos for Illumina® #E6609 (New England
BioLabs). Quality and integrity of the tagged libraries were initially assessed
with the HT DNA HiSens Reagent kit (Perkin Elmer) using a LabChip GX bioanalyser
(Caliper Life Sciences/Perkin Elmer). Tagged libraries were then sized and
quantitated in duplicate (Agilent TapeStation system) using D1000 ScreenTape and
reagents (Agilent). Libraries were normalized, pooled and then clustered using
the HiSeq® 3000/4000 PE Cluster Kit (Illumina). The libraries were imaged
and sequenced on an Illumina HiSeq 4000 sequencer using the HiSeq®
3000/4000 SBS kit (Illumina) at a minimum of 25 million paired-end reads (75
bp/100 bp) per sample.
5
cDNA Library Preparation for Single-Cell RNA-Seq
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