title: GSM8169437: rep2-CocultureTTX; Mus musculus; RNA-Seq
library_construction_protocol: A single-cell suspension from cultured 16T-mGFP SCLC neurospheres was prepared in sterile HBSS immediately before surgery. Animals at postnatal day (P) 21–35 were anaesthetized with 1–4% isoflurane and placed in a stereotactic apparatus. The cranium was exposed via midline incision under aseptic conditions. 70,000 cells in 3 μL sterile HBSS were stereotactically injected into the M2 region of the cortex through a 31-gauge burr hole using a digital pump at infusion rate of 0.4 μL min−1 and 31-gauge Hamilton syringe. Stereotactic coordinates for Thy1-Chr2 animal allografts used were as follows: 0.5 mm lateral to midline, 1.0 mm anterior to bregma, −1.5 mm deep to cortical surface. At the completion of infusion, the syringe needle was allowed to remain in place for a minimum of 2 min, then withdrawn at a rate of 0.875 mm min−1 to minimize backflow of the injected cell suspension. To generate animals with interneuron-specific expression of light-sensitive ion channels, 1 μl of AAV8-Dlx-ChRmine-p2A-mCherry (virus titer=6.9E+12) (a gift from Dr. Karl Deisseroth from Stanford University; Chen et al., 449 2020, Nature Biotech) was unilaterally injected using Hamilton Neurosyringe and 450 Stoelting stereotaxic injector over 5 minutes. These animals then received allografts of 16T-mGFP SCLC cells as described above at 0.8 mm lateral to midline, 1.0 mm anterior to bregma, -1.2 mm deep to the cortical surface. Allografts of 16T-mGFP-ChR2 SCLC cells were performed following the procedure as above with exception for the following alterations: 15,000 cells were injected in 1 μL sterile HBSS 0.8 mm lateral to midline, 1.0 mm anterior to bregma, −1.2 mm deep to cortical surface. For electrophysiology experiments and EM tissue analysis, 16T-mGFP cells were allografted into the CA1 region of the hippocampus at the following coordinates: 1.5 mm lateral to midline, 1.8 mm posterior to bregma, -1.35 mm deep to the cortical surface. The cells were harvested and processed FACS for single cells, then lysed and combined into droplets with barcoded beads which captured the mRNA then used for reverse transcription with the The Chromium Single Cell Gene Expression platform (10x Genomics) per manufacturer's instructions. We then followed the rest of the 10x standard or high-throughput protocols and used the Dual Index Kit TT Set A for library production. 16T SCLC cells were cultured alone or in neuron co-cultures with or without 1 μM TTX for 24 hours per protocol below and harvested in PBS 0.5% BSA, 1mM EDTA (Invitrogen), 1x DNAse (Worthington Biochemical). GFP-negative cells were harvested in parallel to serve as negative control during FACS sorting. Calcein violet (Thermo Fisher) was used to label live cells. GFP+ Calcein+ cells were sorted and collected and processed for scRNA sequencing.