GSM8385815: Fascia_acupuncture_scRNA2; Rattus norvegicus; RNA-Seq
The process of tissue dissociation and cell purification for single-cell RNA sequencing and fascia primary cell extraction was meticulously executed. The fascia was placed in a sterile culture dish containing 10 ml of 1x Dulbecco's Phosphate-Buffered Saline (DPBS; Thermo Fisher, Cat. no. 14190144) on ice to eliminate the residual blood and adipose, followed by mincing on ice.We employed a specially formulated dissociation solution, composed of the collagenase type 4 (sigma-Aldrich, Cat. no. C5138), elastase (sigma-aldrich, Cat. no. e1250), and 10 ug/mL DNase I (Thermo Fisher, Cat. no. en0523) dissolved in PBS with 5% Fetal Bovine Serum (FBS; Thermo Fisher, Cat. no. SV30087.02), to digest the fascia. The fascia was dissociated at 37℃ with a shaking speed of 50 rpm for approximately 40 minutes. We collected the dissociated cells at 20-minute intervals to maximize cell yield and viability.Cell suspensions were filtered by the sequence of nylon cell strainer from 100μm to 40 μm, and residual red blood cells were eliminated using 1X Red Blood Cell Lysis Solution (Thermo Fisher, Cat. no. 00-4333-57). The dissociated cells were then washed with 1x DPBS containing 2% FBS. Cell viability was assessed using 0.4% Trypan blue staining and the Countess® II Automated Cell Counter (Thermo Fisher), all samples of suspension cells were filtered by Dead Cell Removal Kit (Miltenyi Biotec, Cat. No. 130-090-101), and only samples with >95 % living cells were used for next steps.Suspension cells were prepared for 10X library construction and 3' sequencing, water-in-oil solution was prepared by the Chromium™ Single Cell Controller (10X Genomics). The process of tissue dissociation and cell purification for single-cell RNA sequencing and fascia primary cell extraction was meticulously executed. The fascia was placed in a sterile culture dish containing 10 ml of 1x Dulbecco's Phosphate-Buffered Saline (DPBS; Thermo Fisher, Cat. no. 14190144) on ice to eliminate the residual blood and adipose, followed by mincing on ice.We employed a specially formulated dissociation solution, composed of the collagenase type 4 (sigma-Aldrich, Cat. no. C5138), elastase (sigma-aldrich, Cat. no. e1250), and 10 ug/mL DNase I (Thermo Fisher, Cat. no. en0523) dissolved in PBS with 5% Fetal Bovine Serum (FBS; Thermo Fisher, Cat. no. SV30087.02), to digest the fascia. The fascia was dissociated at 37℃ with a shaking speed of 50 rpm for approximately 40 minutes. We collected the dissociated cells at 20-minute intervals to maximize cell yield and viability.Cell suspensions were filtered by the sequence of nylon cell strainer from 100μm to 40 μm, and residual red blood cells were eliminated using 1X Red Blood Cell Lysis Solution (Thermo Fisher, Cat. no. 00-4333-57). The dissociated cells were then washed with 1x DPBS containing 2% FBS. Cell viability was assessed using 0.4% Trypan blue staining and the Countess® II Automated Cell Counter (Thermo Fisher), all samples of suspension cells were filtered by Dead Cell Removal Kit (Miltenyi Biotec, Cat. No. 130-090-101), and only samples with >95 % living cells were used for next steps.Suspension cells were prepared for 10X library construction and 3' sequencing, water-in-oil solution was prepared by the Chromium™ Single Cell Controller (10X Genomics).