title: GSM8073543: TS92_0_replicate_2; Mammaliicoccus sciuri; RNA-Seq
library_construction_protocol: Total RNA was extracted using hot phenol extraction method RNA quality was checked using a 2100 Bioanalyzer with the RNA 6000 Nano kit (Agilent Technologies). cDNA libraries suitable for sequencing were prepared from 1000 ng of total RNA after DNase I treatment (Thermo Fisher). RNAs were treated for rRNA depletion with RiboPOOLs (siTOOLs), except one sample (WT), were fragmented using Mg2+ ions 2 min 45 sec (NEBNext Magnesium RNA fragmentation module) and were treated with T4 PNK for phosphorylation/dephosphorylation and RppH for decapping followed by NEBNext® Multiplex Small RNA Library Prep (New England Biolabs). The number of the PCR cycles was determined at 14 by qPCR and the elongation time was set to 15 sec. Libraries were quantified by QubitTM dsDNA HS Assay Kit 3.0 Fluorometer (ThermoFisher) and quality was checked using 2100 Bioanalyzer with High Sensitivity DNA kit (Agilent Technologies) before pooling. Sequencing of pooled libraries, spiked with 5% PhiX control library, was performed at ~11-15 M reads/sample in single-end mode with 75 nt read length on the NextSeq 500 platform (Illumina).