title: GSM8861822: Parse combined conditions sublibrary 5; Mus musculus; RNA-Seq
library_name: GSM8861822
library_construction_protocol: For single cell RNA sequencing, tibiae were harvested at 4 and 7 days post fracture. Fracture calluses were manually harvested via stereomicroscope dissection from mice at 4 and 7 days post fracture and digested using a shaking waterbath at 37 degrees Celsius for 30 minutes in 10 mL PBS (+Ca/+Mg) supplemented with 2 mg/mL Collagenase P, 2 mg/mL Hyaluronidase, and 200 µg/mL DNase. Periosteum was scraped prior to a 1 hour digestion. scRNAseq (Parse Biosciences pipeline) was used to characterize the transcriptomic differences in early response of Intact and Ischemic WT fracture calluses and CD47-null calluses at 4 and 7 days post fracture and baseline periosteum. For each fracture condition, we sequenced a single biological replicate made up of 8 pooled calluses (equal male/female). Periosteum consisted of 8 pooled limbs (equal male/female). Following enzymatic callus digestion, single cell suspensions were fixed using the Parse fixation kit, frozen down to -80 °C in media containing 1.6% DMSO, and subsequently submitted to the University of Michigan Advanced Genomics Core for sequencing using the Parse Biosciences pipeline. Samples were counted using AO/PI and the LunaFx7 (Logos Bio).Samples were prepped according to manufacturer's instructions (Parse Biosciences Evercode V1). In short, individual transcriptomes are uniquely labeled by passing fixed cells or nuclei through four rounds of barcoding. In each round, pooled cells are randomly distributed into different wells, and transcripts are labeled with well-specific barcodes. Round 1 does reverse transcription of the RNA and adds the first barcode. Rounds 2 and 3 ligate additional barcodes. Round 4 adds the library index, as well as the complete Illumina adapter sequences. cDNA was quantified by Qubit and assessed on TapeStation 2200 (Agilent). Final libraries were quantified by Qubit and assessed on LabChip GX Touch (Revvity).