Qubit rna assay kit

Total RNA was extracted according to manufacturer’s instructions. 1% agarose gels was used for the monitoring of RNA degradation and contamination; the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA) was used for check of RNA purity; Qubit® RNA Assay Kit was used for measurement of RNA concentration; and the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA) was used for assessing of RNA integrity.

RNA libraries were generated and sequenced according to [54 (link)]. RNA was extracted using a RecoverAll™ Total Nucleic Acid Isolation Kit (Invitrogen). RNA concentrations were measured with a Qubit RNA Assay Kit, and an Agilent 2100 bioanalyzer was used to measure the RNA Integrity Number (RIN). The depletion of ribosomal RNA was performed using an RNA Hyper Kit (Roche), and then library concentrations and fragment length distributions were measured with Qubit (Life Technologies) and Agilent Tapestation (Agilent), respectively. The RNA sequencing was performed using an Illumina NextSeq 550 engine for 50 bp single-end reads and 27–39 million raw reads per sample using standard protocol.

Total RNA was isolated from each tissue sample using Trizol reagents (Cat. No. 15596018, Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The integrity of the isolated RNA was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA,USA), and the concentration of RNA was measured using the Qubit® RNA Assay Kit (Cat. No. Q32852, Invitrogen, USA) in a Qubit® 2.0 Fluorometer (Life Technologies, USA).

RNA libraries were generated and sequenced according to Suntsova et al. (24 (link)). RNA was extracted using RecoverAll™ Total Nucleic Acid Isolation Kit (Invitrogen). RNA concentrations were measured with Qubit RNA Assay Kit, and Agilent 2100 bioanalyzer was used to measure RNA Integrity Number (RIN). Depletion of ribosomal RNA was performed using RNA Hyper Kit (Roche), and then library concentrations and fragment length distributions were measured with Qubit (Life Technologies) and Agilent Tapestation (Agilent), respectively. The RNA-seq was performed using Illumina NextSeq 550 engine for 50-bp single-end reads and approximately 30 million raw reads per sample using standard protocol. Single-end sequencing was used because SIA samples were FFPE tissue blocks that typically have a strong degree of RNA degradation. Primary sequencing data quality control was performed with Illumina SAV, and demultiplexing was made according to Suntsova et al. (24 (link)) with Illumina Bcl2fastq2 v 2.17 software.