title: GSM5799929: CRH_Hu852; Homo sapiens; RNA-Seq
library_construction_protocol: For cell collection, 12 µm-thick fresh-frozen sgACC sections were thaw-mounted onto polyethylene naphthalate-membrane slides (Leica Microsystems, Concord, ON, Canada; 11600289) and stored at -80°C. CRH-expressing inhibitory neurons were visualized using FISH labeling with RNAScope Multiplex Fluorescent V2 Assay (Advanced Cell Diagnostics; catalog no. 323100) with some modification. On the day of experiment, slides were taken from -80°C and immediately immersed in chilled 4% PFA diluted in PBS for 5 minutes. Sections were dehydrated by dipping slides serially in 50%, 70%, 100% ethanol and a hydrophobic barrier was drawn around each tissue section.  Sections were treated with hydrogen peroxide for 7 minutes to inactivate endogenous peroxidase and further treated with Protease 3 for 7 minutes at room temperature. After PBS wash, human CRH probe was applied on the section and incubated at 40°C for 1 hour. Excessive probes were removed by rinsing sections twice with wash buffer for several seconds and selective signal amplification was achieved by sequential incubation of Amp 1-3, HRP-C1, TSA Plus Fluorescein (PerkinElmer; catalog no. NEL741E001KT) and HRP blocker. After the final wash, tissue sections were counterstained with Neurotrace red (Thermo Fisher Scientific; catalog no. N21482) and dehydrated with 100% ethanol. ~150 cells were collected from layer 1-3 of gray matter per each subject using a LMD7 system (Leica Microsystems) and processed for RNA extraction using Picopure RNA isolation kit (Thermo Fisher Scientific; catalog no. KIT0204). Sequencing libraries are prepared using the SMARTer Stranded Total RNA-Seq Kit Pico Input Mammalian (Clontech, Mountain View, CA, USA; catalog no. 634413). cDNA fragment size distribution of each library was determined with Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) to check the presence of adapter dimers and gDNA contamination. Libraries were pooled and sequenced using Illumina MiSeq system (Illumina, San Diego, CA, USA) for quality check (QC), and then Illumina NovaSeq 6000 sequencer at Donnelly Sequencing Center at the University of Toronto (ccbr.utoronto.ca/donnelly-sequencing-centre).