GSM6914673: sci 7dpi replicate1 RNAseq; Rattus norvegicus; RNA-Seq
Animals (5 animals for each condition) were anesthetized deeply with pentobarbital sodium, and from the center of the spinal cord injury site, a 5-mm section of the spinal cord was dissected. For extraction of RNA in cells, the tissue was cut and chopped into small pieces, then incubated in 2 ml of collagenase (1 mg/ml in PBS) for 30 min at 37°C and centrifuged at 300 g for 5 min at 4°C. Pelleted cells were collected in 2ml of 0.25% trypsin-EDTA solution and incubated at 37°C for 5 min. Then, digestion by trypsin was stopped by FBS (10%), and the pellet collected by centrifugation was dispersed in PBS with 0.5% BSA. The dispersed cell pellet was strained through a 40-µm cell strainer twice, ensuring there was no large cell cluster exist. To avoid the influence of blood cells, 10 ml of erythrocyte lysate was added to the cell for 10 min, and collect cell suspension by centrifuging. Finally, single-cell suspension was resuspended in PBS for counting. Libraries were prepared according to 10X Genomics Chromium Single Cell 3′ Reagent Kit v3 (10X Genomics) and Single Cell 3' Gel Bead Kit v2 instructions and an Agilent 2100 Expert High Sensitivity DNA Assay was used to assessing the quantity of cDNA. Finally, cDNA libraries were sequenced using an Illumina HiSeqTM 4000 system