Nebnext mrna magnetic isolation module

RNA from 11 cell lines was extracted using the RNeasy Mini Kit (Qiagen, #74104) according to the manufacturer’s protocol. The evaluation of the isolated RNA integrity and quantity was carried out by the Agilent TapeStation system using an RNA ScreenTape (Agilent Technologies, #5067-5576).
For the mRNA library preparation, 4000 ng of total RNA was treated with TurboDNase to remove genomic DNA contamination, (Invitrogen, #AM1907). PolyA RNA was selected from 1000 ng of the purified RNA using NEBNext mRNA magnetic isolation module [New England BioLabs (NEB), #E7490] following the manufacturer’s directions. From the resulting mRNA, strand-specific libraries were created using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB, #E7760). Final libraries were quantified using quantitative polymerase chain reaction and clustered at a molarity of 300 pM. Sequencing was performed on an Illumina NovaSeq 6000 (Illumina) using paired end ×100 cycles v1.0 chemistry, to achieve coverage of 25 million reads per sample.

The construction of cDNA libraries was performed according to a standard protocol using a NEBNext Multiplex Small RNA Library Prep Kit for Illumina (New England Biolabs, UK) for small RNA fraction, NEBNext Ultra II Directional RNA library preparation kit for Illumina (New England Biolabs, Hitchin, UK) and NEBNext mRNA Magnetic Isolation Module (New England Biolabs, UK) for poly(A)+ RNA fraction. For the prepared sequencing libraries, fragment size distribution was analysed using a Bioanalyzer 2100 instrument (Agilent, USA) with an Agilent High Sensitivity DNA Kit (Agilent, USA) and quantification by Qubit DNA HS Assay Kit (Thermo Fisher Scientific, USA) with Qubit 2 fluorometer (Thermo Fisher Scientific, USA). Fragment size ranges between 100 bp to 200 bp and between 250 bp to 700 bp were observed for small RNA and poly(A)+ RNA libraries respectively. Libraries were sequenced on Illumina NextSeq 1500 instrument in 100-base-pair-single-end mode (NextSeq 500/550 High Output v2.5 Kit (Illumina, USA)). The construction of cDNA libraries and massive parallel sequencing were conducted at the Institute of Fundamental Medicine and Biology, Kazan Federal University (Kazan, Russia).

The construction of Illumina cDNA libraries was performed according to a standard protocol using a NEBNext Ultra II Directional RNA library preparation kit (New England Biolabs, Ipswich, UK) and NEBNext mRNA Magnetic Isolation Module (New England Biolabs, UK), as well as massive parallel sequencing on a NextSeq Illumina 1500 platform, at the Institute of Fundamental Medicine and Biology, Kazan Federal University (Kazan, Russia). For the isolation of mRNA, and the fragmentation and priming procedure, 1 μg of the total RNA was used. A NextSeq 500/550 High Output v2.5 Kit (100-nucleotide single-end reads) (Illumina, San Diego, CA, USA) was used. For the prepared sequencing libraries, fragment size distribution was analyzed using a Bioanalyzer 2100 instrument (Agilent, USA) with an Agilent High Sensitivity DNA Kit (Agilent, USA) and quantified using the Qubit 2.0 Fluorometer (Invitrogen, Waltham, MA, USA) with the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, USA). Fragment sizes ranged between 250 bp and 700 bp, with a clear peak at 300 bp.