For the CSU cohort, total RNA was depleted of rRNA using the Ribo-Zero rRNA removal kit, with 1 μg of total RNA used as input for rRNA removal. Sequencing libraries were generated using the TruSeq RNA sample prep kit (Illumina). The libraries were sequenced as 151 bp paired-end reads using an Illumina HiSeq X Ten platform. For the Yale cohort, rRNA was depleted starting from 25–1000 ng of total RNA using the Kapa RNA HyperPrep Kit with RiboErase (KR1351). Indexed libraries that met appropriate cut-offs for both quantity and quality were quantified by qRT-PCR using a commercially available kit (KAPA Biosystems) and insert size distribution was determined with the LabChip GX or Agilent Bioanalyzer. Samples with a yield of ≥0.5 ng/ul were used for sequencing. Samples were run on a combination of Illumina HiSeq 2500, HiSeq 4000, and NovaSeq instruments, and multiplexed using unique dual barcode indexes (to avoid sample contamination and barcode hopping).
Labchip gx
Manufactured by Agilent Technologies
The LabChip GX is a microfluidic-based automated electrophoresis system designed for the analysis of biomolecules. It provides rapid, high-resolution separation and quantification of DNA, RNA, and proteins in a user-friendly platform.
Automatically generated - may contain errors
Lab products found in correlation
Agilent bioanalyzer, by Agilent Technologies (8 mentions)
Hiseq 2500 system, by Illumina (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Rnaeasy mini columns, by Qiagen (3 mentions)
Novaseq, by Illumina (2 mentions)
Bioanalyzer, by Agilent Technologies (2 mentions)
Bioanalyzer gel, by Agilent Technologies (2 mentions)
Truseq rna exome protocol, by Illumina (2 mentions)
Hiseq 4000, by Illumina (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Hiseq x ten, by Illumina (1 mentions)
Truseq rna sample prep kit, by Illumina (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Kapa mrna hyperprep kit, by Roche (1 mentions)
Casava 1.8.2 software suite, by Illumina (1 mentions)
Rneasy mini column, by Qiagen (1 mentions)
12 protocols using labchip gx
1
Comprehensive RNA-seq protocol for diverse samples
2
Strand-specific RNA-seq library preparation
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mRNA was purified from ∼200 ng of total RNA with oligo-dT beads and sheared by incubation at 94°C in the presence of Mg (Kapa mRNA Hyper Prep). Following first-strand synthesis with random primers, second-strand synthesis and A-tailing were performed with dUTP for generating strand-specific sequencing libraries. Adapters with 3′ dTMP overhangs were ligated to library insert fragments. Library amplification was used to amplify fragments carrying the appropriate adapter sequences at both ends. Strands marked with dUTP were not amplified. Indexed libraries that met appropriate cutoffs for the samples passing the size distribution and concentration quality controls were quantified by qRT-PCR using a commercially available kit (KAPA Biosystems), and insert size distribution was determined with the LabChip GX or Agilent Bioanalyzer. Samples with a yield of ≥0.5 ng/µl were used for sequencing.
3
RNA-seq Library Preparation Pipeline
4
RNA Sequencing Library Preparation
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Total RNA quality was determined by estimating the A260/A280 and A260/A230 ratios by nanodrop. RNA integrity was determined by running an Agilent Bioanalyzer gel, which measured the percentage of RNA fragments greater than 200nt for each sample (also called DV200). RNA Seq Library Prep: Following the Illumina TruSeq RNA Exome protocol (Cat. No. RS-301-2001), samples were binned based on DV200 scores. High quality samples had a DV200 > 70%, medium quality samples had a DV200 of 50-70%, low quality samples had a DV200 of 30-50%, and samples with a DV200 < 30% were not used. The RNA was fragmented using divalent cations under elevated temperature at various time periods based on DV200. cDNA was generated from the cleaved RNA fragments using random priming during first and second strand synthesis. Then, sequencing adapters were ligated to the resulting double-stranded cDNA fragments. The coding regions of the transcriptome were captured from this library using sequence-specific probes to create the final library. Indexed libraries that meet appropriate cut-offs for both were quantified by qRT-PCR using a commercially available kit (KAPA Biosystems) and insert size distribution determined with the Perkin Elmer LabChip GX or Agilent Bioanalyzer. Samples with a yield of ≥0.5 ng/ul were used for sequencing.
5
Strand-specific mRNA Sequencing Library Prep
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mRNA was purified from ∼200 ng of total RNA with oligo-dT beads and sheared by incubation at 94°C (Kapa mRNA Hyper Prep). Following first-strand synthesis with random primers, second-strand synthesis was performed with dUTP for generating strand-specific sequencing libraries. The cDNA library was then end repaired and A tailed, adapters were ligated, and second-strand digestion was performed by uracil-DNA-glycosylase. Indexed libraries that met appropriate cutoffs for both were quantified by quantitative RT-PCR using a commercially available kit (KAPA Biosystems) and insert size distributions were determined with the LabChip GX or Agilent Bioanalyzer. Samples with a yield of ≥0.5 ng/µl were used for sequencing.
6
RNA-seq Library Preparation and Analysis
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RNA was extracted from whole cells with the RNeasy Mini kit (Qiagen) according to the manufacturer’s protocol. RNA quality and integrity were determined with an Agilent Bioanalyzer gel with RNA integrity number score higher than 7. RNA-seq libraries were prepared from total RNA with the Kapa mRNA HyperPrep kit (Kapa Biosystems), and library size distributions were determined with the LabChip GX or Agilent Bioanalyzer. Sequencing was performed with Illumina NovaSeq using 100-bp paired-end sequencing RNA-seq at the Yale Center for Genome Analysis. A positive control (prepared bacteriophage PhiX library) provided by Illumina was spiked into every lane at a concentration of 0.3% to monitor sequencing quality in real time. Primary analysis, sample demultiplexing and alignment to the human genome, was performed using Illumina’s CASAVA 1.8.2 software suite. The reads were trimmed for quality using custom scripts. Minimum length accepted was 45 bases. The trimmed reads were then aligned to the mm10 reference genome using Gencode annotation, HISAT2 for alignment, and StringTie for transcript abundance estimation (38 (link), 39 (link)). The generated counts were processed with DESeq2 (40 (link)) in R to determine statistically significantly expressed genes (q < 0.05).
7
RNA-seq Analysis of EPZ-5676 Treatment
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IGROV-1 and SK-OV-3 cells treated with vehicle or 10 µM EPZ-5676 for 48 h were used to prepare total RNA, which was then used for gene expression analysis on an Illumina HiSeq 2500 system. Total RNA was extracted using TRIzol® reagent (Invitrogen) according to the manufacturer’s instructions and then purified on RNAeasy mini columns (Qiagen) according to the manufacturer’s instructions. mRNA was purified from ~500 ng total RNA using oligo-dT beads and then sheared by incubation at 94 °C. Following first-strand synthesis with random primers, second-strand synthesis was performed with dUTP to generate strand-specific sequencing libraries. The cDNA libraries were then end-repaired and A-tailed. Adapters were ligated, and second-strand digestion was performed using Uracil-DNA-Glycosylase. Indexed libraries that met appropriate cut-offs for both were quantified by qRT-PCR using a commercially available kit (KAPA Biosystems, Wilmington, MA, USA). The insert size distribution was determined using LabChip GX or an Agilent Bioanalyzer. Samples with a yield ≥0.5 ng/μl were used for sequencing on the Illumina HiSeq 2500 system. Images generated by the sequencers were converted into nucleotide sequences by the base-calling pipeline RTA 1.18.64.0 and stored in FASTQ format.
8
Transcriptomic Analysis of UBE2T Knockdown
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MCF7 and T47D cells expressing NS or UBE2T shRNA were used to prepare total RNA, which was then used for gene expression analysis on an Illumina HiSeq 2500 system. Total RNA was extracted using TRIzol® reagent (Invitrogen), according to the manufacturer's instructions, and purified on RNeasy mini columns (Qiagen), according to the manufacturer's instructions. mRNA was purified from ∼500 ng total RNA using oligo-dT beads and sheared by incubation at 94°C. Following first-strand synthesis with random primers, second-strand synthesis was performed with dUTP to generate strand-specific sequencing libraries. The cDNA libraries were then end-repaired and A-tailed. Adapters were ligated, and second-strand digestion was performed using uracil–DNA–glycosylase. Indexed libraries that met appropriate cutoffs for both were quantified by qPCR using a commercially available kit (KAPA Biosystems, Wilmington, MA, USA). The insert size distribution was determined using LabChip GX or an Agilent Bioanalyzer. Samples with a yield ≥0.5 ng/μl were used for sequencing on the Illumina HiSeq 2500 system. Images generated by the sequencers were converted into nucleotide sequences by the base-calling pipeline RTA 1.18.64.0 and stored in fastq format.
9
Strand-Specific RNA-Seq Library Preparation
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mRNA was purified from approximately 500 ng of total RNA with oligo-dT beads and sheared by incubation at 94°C. Following first-strand synthesis with random primers, second strand synthesis was performed with dUTP for generating strand-specific sequencing libraries. The cDNA library was then end-repaired, A-tailed, adapters were ligated, and second-strand digestion was performed by Uracil-DNA-Glycosylase. Indexed libraries that met appropriate cut-offs for both were quantified by qRT-PCR using a commercially available kit (KAPA Biosystems) and insert size distribution determined with the LabChip GX or Agilent Bioanalyzer. Samples with a yield of ≥ 0.5 ng/μl were used for sequencing.
10
RNA-Seq of Ovarian Cancer Cell Lines
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Ovarian cancer cell lines (PA-1 and SK-OV3 cells) were treated with BAY-850 (5 μM) and control for 48 h were used to prepare total RNA for gene-expression analysis on an Illumina HiSeq 2500 system. Total RNA was extracted using TRIzol® reagent (Invitrogen) according to the manufacturer’s instructions and purified on RNAeasy mini columns (Qiagen) according to the manufacturer’s instructions. Then, mRNA was purified from approximately 300 ng total RNA using oligo-dT beads and sheared by incubation at 94 °C. Following first-strand synthesis with random primers, second-strand synthesis was performed with dUTP to generate strand-specific libraries. The cDNA libraries were then end-repaired and A-tailed. Adapters were ligated, and second-strand digestion was performed using Uracil-DNA-Glycosylase. Indexed libraries that met appropriate cutoffs for both were quantified by qRT-PCR using a commercially available kit (KAPA Biosystems, Wilmington, MA, USA). The insert-size distribution was determined using LabChip GX or an Agilent Bioanalyzer. Samples with a yield ≥ 0.5 ng/μl were used for sequencing on the Illumina HiSeq 2500 system. Images were converted into nucleotide sequences by the base-calling pipeline RTA 1.18.64.0 and stored in FASTQ format.
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