GSM8270222: femoral condyle, female bipedal mice at 22 weeks of age, scRNAseq; Mus musculus; RNA-Seq
Ten femoral condyle tissue, excluding the growth plate, was isolated from the distal epiphyseal line of hindlimbs of 5 mice in each group at 22 weeks of age. Then the femoral condyles tissues were washed in ice-cold RPMI1640, and dissociated using Collagenase Ⅱ (Sigma, V900892-100MG) and DNase Ⅰ (Sigma 9003-98-9). Cell count and viability was estimated using fluorescence Cell Analyzer (Countstar® Rigel S2) with AO/PI reagent after removal erythrocytes (Solarbio R1010) and then debris and dead cells removal was decided to be performed or not (Miltenyi 130-109-398/130-090-101). Finally fresh cells were washed twice in the RPMI1640 and then resuspended at 1×106 cells per ml in 1×PBS and 0.04% bovine serum albumin. The CD45-positive cells were selected by flow cytometric sorting, and the CD45-positive and negative cells were mixed in a ratio of 1:1 before the examination. Single-cell RNA-Seq libraries were prepared using SeekOne® Digital Droplet Single Cell 3' library preparation kit (SeekGene Catalog No. K00202). Briefly, appropriate number of cells were mixed with reverse transcription reagent and then added to the sample well in SeekOne® chip. Subsequently Barcoded Hydrogel Beads (BHBs) and partitioning oil were dispensed into corresponding wells separately in chip. After emulsion droplet generation reverse transcription were performed at 42℃ for 90 minutes and inactivated at 80℃ for 15 minutes. Next, cDNA was purified from broken droplet and amplified in PCR reaction. The amplified cDNA product was then cleaned, fragmented, end repaired, A-tailed and ligated to sequencing adaptor. Finally, the indexed PCR were performed to amplified the DNA representing 3' polyA part of expressing genes which also contained Cell Barcode and Unique Molecular Index. The indexed sequencing libraries were cleanup with SPRI beads, quantified by quantitative PCR (KAPA Biosystems KK4824) and then sequenced on illumina NovaSeq 6000 with PE150 read length.