RNA was isolated from PEO1 olaparib-sensitive (n=2) and four PEO1 olaparib-resistant clones using RNeasy columns with on-column DNase digest (Qiagen). RNA quality was confirmed using an Agilent Tapestation and all RNA used for library preparation had a RIN>9. Libraries were created using Illumina TruSEQ stranded mRNA library prep (#RS-122-2102). Strand-specific pair-ended libraries were pooled and run on HiSeq4000 (Illumina). Library creation and sequencing were performed at the Genomics Core at the University of Colorado Anschutz Medical Campus. HISAT2 (15 (link)) was used for alignment against GRCh37 version of the human genome. Samples were normalized using TPM (Transcripts per Million) measurement and gene expression using the GRCh37 gene annotation was calculated using home-made scripts. The analysis was performed by the Division of Translational Bioinformatics and Cancer Systems Biology at the University of Colorado School of Medicine. RNA-sequencing has been deposited to NCBI: GSE117765.
On column dnase digest
Manufactured by Qiagen
The On-column DNase digest is a laboratory tool designed to remove DNA contamination from RNA samples during RNA purification. It performs an on-column digestion of DNA using a recombinant DNase enzyme, allowing for the efficient removal of DNA without the need for additional purification steps.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (11 mentions)
Hiseq 4000, by Illumina (3 mentions)
Truseq stranded mrna library prep, by Illumina (3 mentions)
Tapestation, by Agilent Technologies (3 mentions)
Truseq stranded mrna library kit, by Illumina (2 mentions)
Hiseq 4000 system, by Illumina (2 mentions)
Rneasy plus rna extraction kit, by Qiagen (2 mentions)
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
Itaq universal sybr green supermix, by Bio-Rad (2 mentions)
Lc480 quantification software module, by Roche (2 mentions)
Quantstudio 3, by Thermo Fisher Scientific (2 mentions)
Sybr green 1 step iscript, by Bio-Rad (2 mentions)
Rnaprotect, by Qiagen (2 mentions)
Tissue tearor, by Biospec (2 mentions)
β mercaptoethanol, by Qiagen (2 mentions)
Rnaeasy plus mini kit, by Qiagen (2 mentions)
Qscript cdna synthesis kit, by Quantabio (1 mentions)
5 mm stainless steel bead, by Qiagen (1 mentions)
Tissuelyser lt, by Qiagen (1 mentions)
Qiashredder, by Qiagen (1 mentions)
19 protocols using on column dnase digest
1
Transcriptome Profiling of Olaparib-Resistant Ovarian Cancer
2
RNA-Seq Protocol for Differential Gene Expression Analysis
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Three biological replicates were sequenced per condition. Total RNA was extracted from cells using a RNeasy Plus RNA extraction kit (Qiagen, 74136). An on-column DNase digest (Qiagen, 79254) was performed according to the manufacturer’s protocol. RNA-seq libraries were generated using a TruSeq stranded mRNA library kit (Illumina, RS-122-2001) and sequenced by the University of Manchester Genomic Technologies Core Facility on a HiSeq 4000 System (Illumina).
Reads were trimmed using Trimmomatic v0.32 [56 (link)] and aligned to the human genome (GRCh37, hg19, RefSeq transcript annotation) using STAR v2.3.0 [64 (link)]. Gene expression counts were obtained using featureCounts v1.6.2 [62 (link)] and differentially expressed genes were identified using DESeq2 v1.14.1 using FDR < 0.05 [63 (link)]. For results from the lapatinib treatment timecourse, differentially expressed genes were filtered to remove genes in which no timepoint had an FPKM value > 1. Metascape [65 (link)] was used for gene ontology analysis of differentially expressed genes. Ingenuity Pathway Analysis [66 (link)] was used to predict upstream regulators.
Patient tumour samples in the OCCAMS dataset expressing ERBB2 at high levels (ERBB2HIGH) were defined by ERBB2 expression levels greater than the median +2 SD.
Reads were trimmed using Trimmomatic v0.32 [56 (link)] and aligned to the human genome (GRCh37, hg19, RefSeq transcript annotation) using STAR v2.3.0 [64 (link)]. Gene expression counts were obtained using featureCounts v1.6.2 [62 (link)] and differentially expressed genes were identified using DESeq2 v1.14.1 using FDR < 0.05 [63 (link)]. For results from the lapatinib treatment timecourse, differentially expressed genes were filtered to remove genes in which no timepoint had an FPKM value > 1. Metascape [65 (link)] was used for gene ontology analysis of differentially expressed genes. Ingenuity Pathway Analysis [66 (link)] was used to predict upstream regulators.
Patient tumour samples in the OCCAMS dataset expressing ERBB2 at high levels (ERBB2HIGH) were defined by ERBB2 expression levels greater than the median +2 SD.
3
Olaparib-resistant ovarian cancer transcriptome
4
Quantitative PCR Workflow for Gene Expression
5
Retinal RNA Extraction from Rhabdomys
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Rhabdomys pumilio were killed via cervical dislocation, and both eyes were removed and placed in cold sterile PBS. Each retina was then dissected and placed into a separate sterile RNase- and DNase-free 1.5 ml tube containing 0.5 ml of RNAlater and placed on ice. Retina tissue was stored in RNAlater at −20°C until RNA extraction, which was performed using an RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions, with additional on-column DNase digest (Qiagen) to eliminate potential genomic DNA contamination. Tissue was disrupted using mortar and pestle and homogenised with syringe and needle. The optional extra elution step was also undertaken. RNA was immediately used for cDNA synthesis or stored at −80°C. cDNA synthesis was performed using qScript cDNA Synthesis Kit (QuantaBio) according to the manufacturer's instructions, and stored at −20°C until use.
6
RNA Extraction from Snap-Frozen Tissue
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Snap frozen material was disrupted using TissueLyser LT (Qiagen), 5 mm stainless steel beads (Qiagen) and reagent DX (Qiagen). The lysate was further homogenized using QIAshredder (Qiagen) and RNA was extracted using RNeasy mini kit (Qiagen) with on column DNAse digest (Qiagen) and additional washing steps. The quality of the RNA was assessed using an Experion RNA StdSens Analysis Kit (Bio-Rad).
7
Olaparib Resistance RNA Sequencing
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RNA was isolated from PEO1 olaparib-sensitive (n = 2) and four PEO1 olaparib-resistant clones using RNeasy columns with on-column DNase digest (Qiagen). RNA quality was confirmed using an Agilent Tapestation and all RNA used for library preparation had a RIN > 9. Libraries were created using Illumina TruSEQ stranded mRNA library prep (#RS-122-2102). Strand-specific pair-ended libraries were pooled and run on HiSeq4000 (Illumina). Library creation and sequencing were performed at the University of Colorado Genomics Core. HISAT [37 (link)] was used for alignment against GRCh37 version of the human genome. Samples were normalized using transcripts per kilobase million (TPM) measurement and gene expression using the GRCh37 gene annotation was calculated using home-made scripts. Analysis was performed by the Division of Translational Bioinformatics and Cancer Systems Biology at the University of Colorado School of Medicine. Data have been deposited to NCBI GSE117765.
8
RNA Extraction and qPCR Quantification
9
Quantitative Real-Time PCR Protocol
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Total RNA was isolated using a Qiagen RNeasy minikit with on-column DNase digest (Qiagen). First strand cDNA was generated using SuperScript II reverse transcriptase (Invitrogen). Real-time PCR was performed in technical duplicates using Roche LightCycler480 probe master and primers in combination with predesigned monocolor hydrolysis probes of the Roche universal probe library (UPL). For quantification, the Roche LC480 quantification software module was used. Expression levels first were normalized to Gapdh or Tbp expression, followed by normalization to control conditions specific to the individual experiment, as indicated.
10
HDAC and Tumor Suppressor Gene Expression
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RNA was isolated from cells with RNeasy Mini Kit followed by on-column DNAse digest (Qiagen). mRNA expression for HDAC1-11, ARID1A, and TP53 was determined using SYBR green 1-step iScript (Bio-Rad) with Life Technologies QuantStudio 3. β-2-microglobulin (B2M) was used as an internal control. All primer sequences are listed in Supplementary Table 1 .
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