GSM7017261: NT 1; Mus musculus; RNA-Seq
Maxillae were pulverized in a homogenizer, and total RNA was isolated from bones lysates or from cultured cells with TriZol (1000 ul per sample; Thermo Fisher Scientific). RNA purity was detected with a Nanodrop. DNA-free RNA was obtained by using and RNeasy Mini Kit (QIAGEN) with DNase treatment according to the manufacturer's instructions. For quality control of RNA extraction yield, an RNA Screen Tape kit (Agilent Technologies), a D1000 Screen Tape kit (Agilent Technologies), Qubit RNA HS Assay kit (Invitrogen) and a Qubit DNA HS Assay kit (Invitrogen) were used for each specific step. RNA samples from Allograft vs Spon. Healing groups were all passed quality control analysis on a Bioanalyzer 2100 (Agilent Technologies). The 3 biological replicates with the highest RNA were used for mRNA library preparation and bioinformatics analysis as described previously. RNA concentration was measured using Qubit 4 Fluorometer (Thermo Fisher Scientific) and RNA quality was measured using Agilent 2200 Tape Station (Agilent). RNA sequencing libraries were prepared using the CEL-Seq2 protocol, as published by (29) with minor modifications. Instead of single cells as input, 2 ng purified RNA was taken as input for library preparation. The CEL-Seq2 libraries were sequenced on an Illumina NextSeq 550 sequencer (Illumina). RNA measurements, library preparation and sequencing were performed by the Technion Genome Center, Technion, Israel.