Agilent rna 6000 nano microchips

Total RNA was extracted from two million cells with TRIzolate reagent in biological duplicates (originating from two different growths of cells from the same tier of the biobank). For details, see Supplementary Methods. RNA concentration and sample purity were determined using a NanoDrop 1000 instrument (Thermo Fisher Scientific, Waltham, MA, USA). For the analysis of fragment distribution and calculation of RIN values (calculated being at least 9.4; mean: 9.8), RNA samples were loaded to Agilent RNA 6000 Nano microchips (Agilent, Santa Clara, CA, USA). Sequencing libraries were prepared following Illumina’s TruSeq RNA Sample Preparation v2 Guide with poly(A) selection using 1 μg total RNA as the starting material. Indexed libraries were pooled and subjected to single-end sequencing on a NextSeq500 sequencer (Illumina, San Diego, CA, USA) with 50-bp read length. Library preparation, cluster generation, sequencing and base calling were performed at the Genomic Medicine and Bioinformatic Core Facility at the University of Debrecen, Hungary. Demultiplexing was performed using the bcl2fastq Conversion Software (Illumina).

Fresh cell pellets or lyophilized powders containing 3 million cells were carefully resuspended and vortexed for 5 min in 1 ml TRIzolate reagent (UD-Genomed Medical Genomic Technologies Ltd., cat. URN0103). Phase separation was carried out using chloroform (1:5) (Sigma-Aldrich, cat. C2432) and high-speed centrifugation. RNA was precipitated from the aqueous phase for 10 min at room temperature using isopropanol (1:1) (Sigma-Aldrich, cat. I9516). Pellets were washed twice with chilled 75% ethanol (diluted with nuclease-free water from absolute ethanol, VWR International, cat. 20821.296), vacuum-concentrated and redissolved in nuclease-free water (AccuGENE, Lonza, cat. 51200) at 65° C for 10 min. Sample purity was determined using a NanoDrop 1000 instrument (Thermo Fisher Scientific, Waltham, MA, USA), and accurate concentrations were determined using a Qubit RNA HS Assay Kit (Thermo Fisher Scientific, cat. Q32855). Each RNA sample was loaded on Agilent RNA 6000 Nano microchips (Agilent, Santa Clara, CA, USA) according to the manufacturer’s recommendations for the analysis of total RNA fragment distribution and calculation of RIN values.