GSM9462267: Sorted DRG neurons from naive mice; Mus musculus; RNA-Seq
Single-cell suspensions of DRG cells isolated from male and female BALB/cAnNRj mice (n=5) were treated with an FcR blocking solution (No. 130-092-575; Miltenyi Biotec). The cells were subsequently stained with Calcein AM (425201; BioLegend) and LIVE/DEAD™ Fixable Violet Dead Cell Stain (L34955, Invitrogen). The following primary antibodies were used: NRXN2 (BS-11104R, ThermoFischer), PIRT (20990-1-AP, Proteintech) and CD45:BV605 (103139, BioLegend). NRXN2 and PIRT were conjugated using specific labeling kits: PE/R-Phycoerythrin Conjugation Kit - Lightning-Link (ab102918, Abcam) and APC Conjugation Kit - Lightning-Link (ab201807, Abcam) respectively. Staining was performed at 4°C for 30 minutes, after which the cells were washed and resuspended in PBS. Viable, single cells (Calcein⁺, LIVE/DEAD⁻) were then sorted using a BD FACSAria Fusion instrument. The flow cytometry sorting was performed at the Biomedicum Flow cytometry Core facility that receives funding from the Infrastructure Board at Karolinska Institutet. Flow cytometry data was analyzed using FlowJo 10 software. Single NRXN2+PIRT+AF+ cells were sorted into 0.3uL Smart-seq3xpress lysis buffer and processed according to previously published protocol (ref 9) with the following project specific alterfications; Preamplification was carried out using 13 cycles of PCR, followed by tagmentation of diluted cDNA using 0.005uL TDE1 (Illumina, USA) per well. Finally, the tagmented libraries were amplified using cell specific index primers for 14 cycles of PCR. Sequencing ready Smart-Seq3xpress libraries were converted to circular ssDNA libraries using the MGIEasy Universal Library Conversion kit App-A (MGI) before sequenced on MGI DNBSEQ G400 platform using universally compatible (App-D) sequencing reagents according to the manufacturer's instructions.