title: RNAseq of BM-MSC: Wall Shear Stress of 8 dyne rep 1
design: Aliquots of 1 million cells were pelleted and snap-frozen in LN2 and submitted to Cancer Genomics Center core facility at The University of Texas Health Science Center at Houston (CPRIT RP180734). Total RNA was exacted by RNeasy Mini Kit (Qiagen, Hilden, Germany) and quality-checked using Agilent RNA 6000 Pico kit by Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, USA). All samples used in this study had an RNA integrity number greater than 7 and were subsequently used for library preparation. rRNA of 400ng total RNA were depleted with NEBNext rRNA Depletion Kit (New England Biolabs, Ipswich, MA) following the manufacturers instructions. The RNAs with more than 70nts were selected for preparation with NEBNext Ultra II Directional RNA Library Prep Kit for ILLumina (New England Biolabs, Ipswich, MA) and NEBNext Multiplex Oligos for illumina (New England Biolabs) following the manufacturers instructions. The quality of the final libraries was examined using Agilent High Sensitive DNA Kit by Agilent Bioanalyzer 2100 (Agilent Technologies), and the library concentrations were determined by qPCR using Collibri Library Quantification kit (Thermo Fisher Scientific). The libraries were pooled evenly and analyzed using paired-end 75-cycle sequencing on an Illumina NextSeq 550 System (Illumina, Inc., San Diego, CA, USA) using High Output Kit v2.5 (Illumina, Inc.).