GSM9758632: AAV infected, Hopx knock down; Mus musculus; RNA-Seq
Aldh1l1CreERT2;Rosa26-LSL-tdTomato mice were injected in the hippocampus with Cre-dependent AAV-TIGRi-Hopx carrying an EGFP reporter. Tamoxifen was administered to induce Cre-mediated recombination and tdTomato lineage labeling in astrocyte-lineage cells. At 3 weeks after tamoxifen induction, mice were sacrificed, and hippocampal tissues were rapidly dissected. The dissected hippocampi were dissociated into single-cell suspensions by enzymatic and mechanical dissociation. The resulting cell suspensions were filtered to remove debris and cell aggregates. Fluorescence-activated cell sorting was then performed to enrich tdTomato-positive/EGFP-positive cells, corresponding to lineage-labeled and AAV-transduced astrocyte-lineage cells. Sorted cells were collected and immediately processed for single-cell RNA-seq library preparation. Single-cell RNA-seq sequencing With the use of a Chromium Single Cell 3' GEM, Library & Gel Bead Kit v3.1 (10x Genomics, PN-1000268), the SIC single-cell suspension (700-1200 living cells per ul as determined by the CellDrop FL Cell Counter) was loaded onto a Chromium Single Cell Chip (Chromium Single Cell G Chip Kit, 10x Genomics, PN-1000120) according to the manufacturer's instructions, for co-encapsulation with barcoded Gel Beads at a target capture rate of $6000 individual cells per sample. We lysed captured cells and released RNA was barcoded through reverse-transcription in individual single-cell gel beads in the emulsion (GEMS). In each droplet, complementary DNA (cDNA) was generated and amplified through reverse-transcription on a T100 PCR Thermal Cycler (Bio Rad) at 53 C for 45 min, followed by 85 C for 5 min and a hold at 4 C. Then, cDNA concentration and quality were assessed using Qubit Fluorometer (Thermo Scientific) and bioanalyzer 2100 (Agilent), respectively. According to the manufacturer's introduction, scRNA-seq libraries were constructed and sequenced to a depth of $40000 reads per cell on illumina platform by Annoroad Gene Technology Co., Ltd.