Bioanalyzer rna 6000 nano assay

Total RNA (500 ng) was used for library preparation. RNA quality control was assessed with the Bioanalyzer RNA 6000 Nano assay on the 2100 Bioanalyzer system (Agilent technologies, Mississauga, ON, Canada), and all samples had an RNA integrity number (RIN) above eight. Library preparation was carried out with the KAPA mRNA-Seq HyperPrep kit (KAPA, Cat no. KK8581). Ligation was made with Illumina dual-index UMI, and 10 PCR cycles were required to amplify cDNA libraries. Libraries were quantified by QuBit and BioAnalyzer DNA1000. All libraries were diluted to 10 nM and normalized by qPCR using the KAPA library quantification kit (KAPA; Cat no. KK4973). Libraries were pooled to equimolar concentrations. Three biological replicates were generated. Sequencing was performed with the Illumina Nextseq500 using the Nextseq High Output 75 (1 × 75 bp) cycles kit. Around 15–20 M single-end PF reads were generated per sample. Library preparation and sequencing was performed at the Genomic Platform of the Institute for Research in Immunology and Cancer (IRIC, Montreal, QC, Canada).

In order to prepare the libraries, total RNA (500 ng) was isolated from HL60 cell cultures. All samples had an RNA integrity number (RIN) above eight when RNA quality control was evaluated using the Bioanalyzer RNA 6000 Nano assay on the 2100 Bioanalyzer system (Agilent Technologies, Mississauga, ON, Canada). Using the KAPA mRNA-Seq HyperPrep kit (KAPA, Cat no. KK8581), the libraries were prepared. Illumina dual-index UMI was used for ligation, and cDNA libraries were required to be amplified using 10 PCR cycles. By using the QuBit and BioAnalyzer DNA1000, libraries were quantified. All libraries were diluted to 10 nM, and the KAPA library quantification kit was used for qPCR normalization (KAPA; Cat no. KK4973). Libraries were pooled to equimolar concentrations. There were three biological replicates.

RNA from 1 million clonal NALM‐6 cells of the indicated genotypes was extracted using the QIAGEN Mini RNeasy kit according to the manufacturer's protocol. Sample purity was assessed by nanodrop using 260/280 nm and 260/230 nm ratios. Total RNA was quantified by QuBit (ABI) and 1 µg of total RNA was used for library preparation. RNA quality control was assessed with the Bioanalyzer RNA 6000 Nano assay on the 2100 Bioanalyzer system (Agilent technologies) and all samples had a RIN of 10. Library preparation was performed with the KAPA mRNAseq Hyperprep kit (KAPA, #KK8581). Libraries were quantified by QuBit and BioAnalyzer and diluted to 10 nM before normalization by qPCR using the KAPA library quantification kit (KAPA; #KK4973). Libraries were then pooled to equimolar concentration. Sequencing was performed with the Illumina Nextseq500 on half a flowcell of Nextseq 75 cycles High Output v2 using 2.8 pM of the pooled libraries. Around 20 million single‐end PF reads were generated per sample. Analysis of RNA‐seq reads is described further in Appendix S1.

For gene profiling human ESC, pooled samples from differentiation batches of human ESC‐derived spheroids and human islets from two different preparations were sampled. Total RNA was extracted using Isolate II RNA Mini Kit (Bioline; BIO‐52072). Concentration of RNA was analyzed using nanodrop. The precise quantity and quality of RNA were determined using Bioanalyzer RNA 6000 Nano assay (Agilent; 5067‐1511). The RNA Integrity Number value for the RNA samples were between 9.9 and 10. For the gene profiling of 50 selected genes, nCounter GX Custom CodeSets (NanoString; 116000001) was used. For each sample, 50 ng of RNA was hybridized with Reporter and Capture ProbeSet in a volume of 15 μL. Hybridization was carried at 65°C for 18 hours followed by a ramp down to 4°C. The hybridization samples were then loaded on nCounter SPRINT Cartridge (nanoString; 100 078) and run on nCounter SPRINT profiler. Data were analyzed using nSolver. Recommended quality control was performed. Background was subtracted using spiked negative controls. Absolute count of RNA was further normalized using the POLR2A housekeeping gene and plotted as heat map using GraphPad prism.

Total RNA (500 ng) was used for library preparation. RNA quality control was assessed with the Bioanalyzer RNA 6000 Nano assay on the 2100 Bioanalyzer system (Agilent technologies), and all samples had a RIN above 8. Library preparation was carried out with the KAPA mRNAseq Hyperprep kit (KAPA, Cat no. KK8581). Ligation was made with Illumina dual-index UMI (IDT), and 10 PCR cycles were required to amplify cDNA libraries. Libraries were quantified by QuBit and BioAnalyzer DNA1000. All libraries were diluted to 10 nM and normalized by qPCR using the KAPA library quantification kit (KAPA; Cat no. KK4973). Libraries were pooled to equimolar concentrations. Three biological replicates were generated. Sequencing was performed with the Illumina Nextseq500 using the Nextseq High Output 75 (1 × 75 bp) cycles kit. Around 15–20 M single-end PF reads were generated per sample. Library preparation and sequencing was performed at the Genomic Platform of the Institute for Research in Immunology and Cancer (IRIC) (Montreal, QC, Canada).

Mice were euthanized using a lethal dose of ketamine (Bimeda-MTC)/xylazine (Bayer Healthcare). Hippocampi from unoperated or pump-implanted mice were dissected and immediately flash frozen on dry ice. Total RNA was extracted with TRIzol (Invitrogen) and chloroform (Invitrogen). RNA was then purified with the RNeasy mini kit (Qiagen) according to manufacturer’s instructions. 400 ng of total RNA was used for library preparation. RNA quality control was assessed with the Bioanalyzer RNA 6000 Nano assay on the 2100 Bioanalyzer system (Agilent technologies) and all samples had a RIN above 8. Library preparation was done with the KAPA mRNAseq Hyperprep kit (KAPA, Cat no. KK8581). Ligation was made with 38 nM final concentration of Illumina dual-index UMI (IDT) and 11 PCR cycles was required to amplify cDNA libraries. Libraries were quantified by QuBit and BioAnalyzer DNA1000. All libraries were diluted to 10 nM and normalized by qPCR using the KAPA library quantification kit (KAPA; Cat no. KK4973). Libraries were pooled to equimolar concentration. Sequencing was performed with the Illumina Nextseq500 using the Nextseq High Output 75 cycles kit using 2.6 pM of the pooled libraries. 20–30M single-end PF reads were generated per sample. Library preparation and sequencing was made at the Institute for Research in Immunology and Cancer’s Genomics Platform (IRIC).