Illumina HiSeq 2000 sequencing; Total RNA-sequencing Detects Transcriptome Differences in Mouse Preosteoblast Response to Bulk Nanostructured Titanium
Total RNA-sequencing Detects Transcriptome Differences in Mouse Preosteoblast Response to Bulk Nanostructured Titanium
Sterile Ti discs were arrayed in an untreated 24-well cell culture plate using a block design, with 12 coarse Grain and 12 ultrafine grain disks.  Wells were re-fed after 24 hours. After 72 hours Ti discs were transferred to new plates for standard cell harvesting protocols.  The cells from four wells were consolidated into one sample resulting in multiple biological replicates for each sample. Substrate for growth: Commercially pure Grade 4 titanium Carpenter Technology Corporation (Reading, Pennsylvania), As-received  RNA was extracted with a Direct-zol RNA Mini Prep kit using the standard protocol (Zymo Research Corp., R2050) and included the treatment of the RNA with DNase I to eliminate genomic DNA. RNA concentrations and quality were determined using a Nanodrop 2000 UV-Vis Spectrophotometer (Thermo Scientific). RNA concentrations were confirmed with the Qubit RNA HS assay kit (ThermoFisher Scientific, Q32852). RNA size and integrity was measured using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).   Ribosomal RNA subtraction was done using the Ribo-Zero® Gold rRNA Removal Kit (Illumina, San Diego). Next generation sequencing libraries preparation utilized an Illumina TruSeq™ RNA Sample Prep Kit v2 (Illumina, San Diego). Unique indexes were added to each sample during library construction.