Nextflex small rna seq kit v3

RNA for sRNA-seq was isolated using miRVana total RNA isolation, following the protocol, (ThermoFisher, #AM1560). sRNA-seq libraries were randomly prepared from 400 ng of RNA using the NEXTFLEX Small RNA-Seq Kit v3 (PerkinElmer, #NOVA-5132-05) using 16 PCR cycles and using the thermal settings as recommended in the protocol. Ten synthetic calibrator RNAs were mixed with the input RNA during the first ligation step as previously described [24 (link)]. After library preparation, sRNAs quality and size were evaluated using Eukaryote 4 total RNA pico assay on the 2100 Bioanalyzer (Agilent Technologies) and sRNA-fragments with size larger than 140 nts (adaptors are 140 nts) and shorter than 413 nts (longest detected fragment) were excised and included in the sequencing.
Bioanalyzer result is presented in Supplementary Fig. 1C. The sequencing libraries were sequenced on the NextSeq 500 System from Illumina.
mRNA-seq was performed on the same patient samples and these data can be accessed in the web application together with the sRNA data. The methods and workflow for the sequencing and analysis of mRNA data are published and previously described [25 (link)].

Small RNA libraries were prepared using NEXTflex Small RNA-Seq Kit v3 (PerkinElmer) according to the manufacturer’s instructions with the following approaches: adapters diluted 1:4, 3’ ligation performed for 18h, samples amplified for 23 PCR cycles, then gel-free size selected according to optional step H1 in the instructions. Libraries were size selected to 140–170 bp using the SAGE Pippin Prep system and repurified by gel-free size selection H1. Sequencing of 1 x 75 bp was performed with an Illumina NextSeq high output kit, resulting in 81,250,738 total reads.