Kapa mrna hyperprep kit v4.17

Libraries were prepared by the Van Andel Genomics Core from 500 ng of total RNA using the KAPA mRNA HyperPrep kit (v4.17) (Kapa Biosystems, Wilmington, MA, USA). RNA was sheared to 300–400 bp. Prior to PCR amplification, cDNA fragments were ligated to Bioo Scientific NEXTflex dual indexing adapters (Bioo Scientific, Austin, TX, USA). Quality and quantity of the finished libraries were assessed using a combination of Agilent DNA High Sensitivity chip (Agilent Technologies, Inc, Santa Clara, CA, USA.), QuantiFluor^® dsDNA System (Promega Corp., Madison, WI, USA), and Kapa Illumina Library Quantification qPCR assays (Kapa Biosystems, Wilmington, MA, USA). Individually indexed libraries were pooled and 75 bp, single-end sequencing was performed on a NextSeq500 sequencer using a 75-cycle high output kit (v2) (Illumina Inc., San Diego, CA, USA) and each library was sequenced to an average raw depth of 35 M reads. Base-calling was done by Illumina NextSeq Control Software (NCS) v2.0 and the output of NCS was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v1.9.0.

Libraries were prepared by the Van Andel Genomics Core from 500 ng of total RNA using the KAPA mRNA Hyperprep kit (v4.17) (Kapa Biosystems, Wilmington, MA, USA). RNA was sheared to 300–400 bp. Prior to PCR amplification, cDNA fragments were ligated to IDT Illumina UDI dual Indexed adapters (Illumina Inc, San Diego, CA, USA). Quality and quantity of the finished libraries were assessed using a combination of Agilent DNF-474 HS fragment kit (Agilent Technologies, Inc.) using a Fragment Analyzer (Agilent Technologies, Inc.), and QuantiFluor^® dsDNA System (Promega Corp., Madison, WI, USA). Individually indexed libraries were pooled and 50 bp, paired end sequencing was performed on an Illumina NovaSeq6000 sequencer using an S2, 100 bp sequencing kit (Illumina Inc., San Diego, CA, USA) to an average depth of 50M reads per sample. Base calling was conducted by Illumina RTA3 and output of NCS was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v1.9.0.