title: GSM8098612: Ppargc1a mutant batch 2; Mus musculus; RNA-Seq
library_construction_protocol: Preparation of single-cell suspension. Lhx6-Cre;Ppargc1a+/+ and Lhx6-Cre;Ppargc1aF/F animals, injected in both hemispheres with AAV8-hSyn-dio-tdTomato viruses at P1, were processed separately in three control and three mutant batches. For each animal, the somatosensory cortex of both hemispheres was dissociated into a single-cell suspension. Mice were deeply anesthetized with sodium pentobarbital by intraperitoneal injection and transcardially perfused with ice-cold oxygenated cutting solution (87mM NaCl, 2.5mM KCl, 1.25mM NaH2PO4, 26 mM NaHCO3, 75mM sucrose, 20mM D-glucose, 0.5mM CaCl2*2H2O, 4mM MgSO4*7H2O). The brain was extracted and incubated for three minutes in an ice-cold oxygenated cutting solution. The somatosensory cortex was then dissected into chunks in icy oxygenated cutting solution, and the tissue was dissociated using the Papain Dissociation System (Worthington #LK003160). After 30 minutes of incubation at 37°C with papain solution, the tissue chunks were washed with an oxygenated cutting solution and were manually and slowly dissociated using a 10mL pipette and a 1mL pipette tip. All solutions used during this process were oxygenated for at least 10 minutes with 95% O2 and 5% CO2. After papain incubation, all steps were performed on ice and using low retention filter tips (VWR). Oxygenation and a short time of dissection are crucial for the viability of the cells in suspension. After dissociation, small tissue chunks may remain, and these were discarded by filtering through a 100µm cell strainer (Fisher Scientific #11587522). The debris was removed by overlaying the single-cell suspension on top of a 10% Percoll solution (Sigma #P4937, diluted in oxygenated cutting solution and 1.5M NaCl). After a 10-min 500g centrifugation at 4°C, the pelleted single-cells were resuspended in cutting solution with 2% BSA, filtered through a 70µm cell strainer (Fisher Scientific #11597522), and transferred into a FACS tube (Falcon #352063) precoated with 3% BSA overnight. Fluorescence-activated cell sorting. Samples were sorted at 4°C on BD FACS Aria II or Aria III Cell sorters for no longer than 40 min and using a 130 µm nozzle to increase cell viability. DAPI was added to the single-cell suspension to discard dead cells. Viable tdTomato+ single cells were collected into 1 ml of cutting solution with 2% BSA in LoBind tubes (SLS #Z666505) precoated with 3% BSA overnight. Collection tubes were centrifugated for 5 min at 4°C at 500 g, and the supernatant was discarded until 45 µl were left in the tube. Cells were resuspended, and cell number and viability were assessed using Trypan Blue and a hemacytometer for cell counts. We only carried out single-cell RNA-sequencing in experiments with viability above 60%. Between 3,000-5,000 cells were loaded for each sample onto a 10X Genomic single-cell chip/machine for single-cell capture and cDNA library preparation. RNA-seq was performed in an Illumina NextSeq 2000 platform. Library construction was performed according to the manufacter's instructions (single cell 3' v3.1protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-AmRNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an IluminaTruSeq Read 1primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR. The amplified cDNA was enzymatically fragmentedand appropriatesize fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction P5,P7,i7 and i5sample indexandIluminaTruSeqRead2 primer sequence were addedvia end repair, A-tailing, adaptor ligation and PCR.