GSM5743383: LCR1_KO scRNA; Mus musculus; RNA-Seq
BM progenitors (Live,CD45+, Lin-, CD127+) were obtained by pooling LCR1+/+ or LCR1-/- animals (same number of males and females; between 8 and 12 weeks old). After cell sorting cells were washed two times with PBS containing 0.01% of Bovine Serum Albumin (BSA, Sigma #A8806) cell viability and cell counts were assessed using an hemacytometer and a trypan blue staining. Libraries were constructed using Chromium™ Controller and Chromium™ Single Cell 3' Library & Gel Bead Kit v3.1 (10x Genomics) according to the manufacturer's protocol for 10000 cells recovery. Briefly, cellular suspension was added to the master mix containing RT Reagent B, Template Switch Oligo, Reducing Agent B and RT Enzyme C. 70mL of master mix with cell suspension was then loaded to the Row 1, while Row 2 and Row 3 were filled with 50mL of Gel Beads 45mL of Partitioning Oil respectively. After run completion, 100mL of GEM mix was used for reverse transcription and was performed using a C1000 Touch Thermal cycler (Bio-Rad) at the following conditions: 53°C for 45 min, 85°C for 5 min, held at 4°C. Post GEM-RT cleanup was performed with DynaBeads MyOne Silane Beads (Thermo Fisher Scientific). cDNA was amplified using C1000 Touch Thermal cycler at the following conditions: 98°C for 3 min, 12 cycles of (90°C for 15 s, 67°C for 20 s and 72°C for 1 min), 72°C for 1 min, held 4°C. Amplified cDNA was cleaned with the SPRIselect Reagent Kit (Beckman Coulter) and quality was assessed using a TapeStation (Agilent). Libraries were constructed following the manufacturer's protocol and sequenced with a targeted sequencing depth of 20.000 read pairs per cell on pair-end mode on either a NextSeq 500/550 or a NovaSeq with recommended loading concentration (1.8pM and 300pM for NextSeq and NovaSeq respectively).