title: GSM5893418: siS6K1_sgS6K2_rep1; Homo sapiens; RNA-Seq
library_construction_protocol: RNA was extracted from the transfected LN229 cell line using TRIzol (Thermo fisher, Australia; cat 15596026) according to the manufacturer's instructions. RNA samples were treated with 2 Units/μL of rDNase I enzyme for 10× DNase I Buffer and incubated at 37 °C for 30 min. The reaction was inactivated by adding DNase Inactivation Reagent. Directional poly A RNA-sequencing was carried out by the Genomics, Epigenomics and Sequencing Core at the University of Cincinnati using standard protocols as established previously (PMID: 31120332 and 31420676). Briefly, total RNA was analyzed to check the quality with the help of Bioanalyzer (Agilent, Santa Clara, CA). NEBNext Poly(A) mRNA Magnetic Isolation Module (New England BioLabs, Ipswich, MA) was used to isolate poly A RNA meant for library preparation. RNA used was 1 µg of good quality as input. Next step was to enrich the polyA RNA with SMARTer Apollo automated NGS library prep system (Takara Bio USA, Mountain View, CA). Library preparation was performed by NEBNext Ultra II Directional RNA Library Prep kit (New England BioLabs) under PCR cycle number of 8. Once the library QC and quantification was done with real-time qPCR (NEBNext Library Quant Kit, New England BioLabs), individually numbered libraries were sequenced by NextSeq 550 sequencer (Illumina, San Diego, CA). Sequencing setting was kept as single read 1x85 bp.