title: GSM3929741: AV_DiI.rs_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 then microdissected and the tissue dissociated. Fluorescent neurons were manually sorted, washed, aspirated into a small volume and lysed with 47 μL XB lysis buffer (PicoPure, Thermo Fisher), incubated for 5 min at 42ºC and stored at -80ºC. Upon thawing, total RNA was extracted using the PicoPure kit (KIT0204, Thermo Fisher) and External RNA Controls Consortium (ERCC) spike-ins added at 1: 10^5 dilution. cDNA was synthesized and amplified using the Ovation RNA-seq v2 kit (NuGEN), yielding 4-8 µg of cDNA, and sheared using a Covaris E220 sonicator. Libraries were generated using the Ovation rapid DR multiplexing kit (NuGEN), quantified by qPCR using Kapa Library Quantification for Illumina (KK4854, Kapa Biosystems), and sequenced on a HiSeq 2500 flow cell (Illumina).
attributes.tag: GEO Accession
attributes.value: GSM3929741