GSM8502832: RK 243; Homo sapiens; RNA-Seq
Bone marrow (BM) was collected from the proximal femur during THA or from the distal femur during TKA. During these procedures, bone marrow was extruded and collected into syringes containing heparin (2,500 IU/mL heparin per 10 mL of bone marrow). The bone marrow preparations were further diluted fourfold with phosphate-buffered saline without Ca2+, Mg2+ (PBS). Samples were filtered through a 100-µm cell strainer to remove bone debris and then slowly applied to the density gradient material using a ratio of 1 volume of Biocoll (Biochrom, Berlin, Germany, L6115; ρ = 1.077 g/ml) to 2 Volumes of diluted bone marrow in 50 ml conical centrifuge tubes were used. After centrifugation at 500 × g for 30 min at room temperature without braking, mononuclear cells were collected from the density gradient. The mononuclear cells were washed once with PBS, followed by centrifugation at 500 × g for 5 min at room temperature with brakes. The cell pellet was resuspended in MSC-GM growth medium (DMEM with 1 g/L D-glucose, with 3.7 g/L NaHCO3, with L-alanyl-L-gutamine (Sigma #FG0415) supplemented with 10% FCS, 25 mM, resuspended HEPES, 100,000 U/L penicillin, 100 mg/L streptomycin, 2 mg/L FGF-2). Cells were seeded into tissue culture plastic bottles at a density of 500,000 mononuclear cells per cm2. The cells were incubated at 37 °C and 5% CO2. The next day, the medium was changed to remove nonadherent cells. For RNA-Seq, MSCs were cultured until reaching passage 3 (medium changed twice per week; trypsin was used for subculture). RNA was extracted from the BM-MCS using Trizol reagent (Invitrogen: 1 ml per 25 cm2 culture surface). The lysate was stored at −80°C until analysis. After thawing, 1/10 volume of 1-bromo-3-chloropropane was added and mixed by inverting. After incubation for 5 minutes at room temperature, the suspension was centrifuged for 15 minutes at 20,000 × g at 4 °C. The clear, colorless upper phase containing the RNA was transferred to a new tube and mixed gently with 0.6 volume of isopropanol. After a short incubation of 5 minutes, the suspension was centrifuged for 30 minutes at 20,000 × g at 4 °C. The resulting pellet was washed twice with ice-cold 80% ethanol (20,000 × g; 4 °C; 15 min). After drying, the pellet was resuspended in RNase-free water and incubated at 58 °C for 10 minutes. Library generation, quality control, and quantification:  300ng of total RNA per sample were utilized as input for mRNA enrichment procedure with 'NEBNext® Poly(A) mRNA Magnetic Isolation Module' (E7490L; New England Biolabs) followed by stranded cDNA library generation using 'NEBNext® Ultra II Directional RNA Library Prep Kit for Illumina' (E7760L; New England Biolabs). All steps were performed as recommended in user manualE7760 (Version 1.0_02-2017; NEB) except that all reactions were downscaled to 2/3 of initial volumes. cDNA libraries were barcoded by dual indexing approach, using 'NEBNext Multiplex Oligos for Illumina – 96 Unique Dual Index Primer Pairs' (6440S; New England Biolabs). All generated cDNA libraries were amplified with 8 of final pcr. One additional purification step was introduced at the end of the standard procedure, using 1.2x 'Agencourt® AMPure® XP Beads' (#A63881; Beckman Coulter, Inc.). Fragment length distribution of individual libraries was monitored using 'Bioanalyzer High Sensitivity DNA Assay' (5067-4626; Agilent Technologies). Quantification of libraries was performed by use of the 'Qubit® dsDNA HS Assay Kit' (Q32854; ThermoFisher Scientific). Library denaturation and Sequencing run: Equal molar amounts of individually barcoded libraries were pooled for, in total, 2 sequencing runs in which each analyzed library constituted around 7.8% of overall flowcell / run capacity. The library pool was denatured with NaOH and was finally diluted to 1.8pM according to the Denature and Dilute Libraries Guide (Document # 15048776 v02; Illumina). 1.3ml of the denatured pool was loaded on an Illumina NextSeq 550 sequencer using a High Output Flowcell (400M cluster) for single reads (20024906; Illumina). Sequencing was performed with the following settings: Sequence reads 1 and 2 with 38 bases each; Index reads 1 and 2 with 8 bases each.