GSM8689613: WT, BMDM, Saa3 treated, scRNAseq (ABCD); Mus musculus; RNA-Seq
After sacrificing the mouse and disinfecting it with 70% ethanol, expose the hind limb muscles by pulling down the skin. Remove the main muscles near the lower limb and carefully isolate the femur and tibia. Place the bones into a petri dish containing ice-cold HBSS with 1% P/S/A and remove any excess muscle. Cut off the bone ends and transfer the bones into a 0.2ml PCR tube with a small hole at its base, placed inside a 1.5 ml tube. Centrifuge at 4000g in 4 °C for 30 secs to collect the bone marrow cells, then resuspend them in culture medium. Filter the cell suspension through a 70 μm strainer to remove clumps, centrifuge again to discard the supernatant, and finally resuspend the cell pellet in bone marrow culture medium. The split-pool barcoding process, following the Evercode™ WT v2 protocol, consisted of three rounds. In Round 1, cells were distributed into wells, and sample-specific barcodes were applied to the fixed cells using an in-cell reverse transcription (RT) reaction. Cells from all wells were then pooled together. In Round 2, cells were redistributed across a plate, and an in-cell ligation was performed to append the second barcode. The cells from each well were again pooled together. In Round 3, cells were split across a plate, and a third barcode was appended with another in-cell ligation. After pooling cells from each well, the pooled cells were divided across multiple sublibraries. The cells were then lysed, and the library was fragmented, end-repaired, and went through A-tailing followed by the ligation of the sequencing adaptor. The library was then labeled with a fourth sublibrary-specific barcode. The quality of dsDNA libraries was analyzed using the High Sensitivity D5000 ScreenTape Kit (Agilent) and concentrations were assessed with the Qubit dsDNA HS Kit (Thermo Fisher Scientific). Libraries were sequenced on a NovaSeq X Plus (Illumina) with 10% PhiX.