title: GSM3929796: Plate_3; Mus musculus; RNA-Seq
library_construction_protocol: Retrogradely labelled cells were collected as previously described (Hempel et al., Nat Protoc 2007; (Hempel et al., Nat Protoc 2007, PMID 18007629; Sugino et al., eLife 2019, PMID 30977723). Coronal slices (200-300 μm) were cut and incubated for 1 hour with pronases and neural activity blockers in artificial cerebrospinal fluid (ACSF). Thalamic areas were microdissected and the tissue dissociated. Fluorescent neurons were manually sorted, washed, and collected into 8-well strips containing 3 µL Smart-seq2 lysis buffer and frozen at -80ºC. Samples for single-cell RNAseq were prepared as described previously (Cembrowski et al., Cell 2018, PMID 29681453). Upon thawing, cells were lysed and digested with Proteinase K, and External RNA Controls Consortium (ERCC) spike-ins added at 1:10^7. cDNA was synthesized using the Maxima H Minus Reverse Transcriptase (Thermo Fisher Scientific) and amplified using the HiFi PCR kit (Kapa Biosystems). Samples were then pooled across strips and purified with Ampure XP beads (Beckman Coulter). These pooled strips were then combined to create a plate-level cDNA pool, and concentration was determined using Qubit High-Sensitivity DNA kit (Thermo Fisher Scientific). Tagmentation and library preparation using 600 pg cDNA from each plate of cells were performed with a modified Nextera XT (Illumina) protocol (Soumillon et al., 2014, bioRxiv 003236), but with the P5NEXTPT5 primer and tagmentation time extended to 15 minutes. Libraries were then purified following the Nextera XT protocol and quantified by qPCR using Kapa Library Quantification (Kapa Biosystems). 6-10 plates were run on a NextSeq 550 flow cell. Read 1 contains the cell barcode and unique molecular identifier (UMI). Read 2 contains a cDNA fragment from the 3' end of the transcript.