GSM5745725: 3day_FGF21_KLB_BLA; Mus musculus; RNA-Seq
The method used here for generating single cell suspensions was modified from a previously published protocol (Wu et al., 2017). Individual mice were quickly decapitated, the brain was immediately extracted and placed in fresh ice-cold artificial cerebrospinal fluid (aCSF) containing 124 mM NaCl, 2.5 mM KCl, 1.2 mM NaH2PO4, 24 mM NaHCO3, 5 mM HEPES, 13 mM glucose, 2 mM MgSO4, and 2 mM CaCl2, bubbled with a carbogen gas (95% O2 and 5% CO2) and with a pH of 7.3 - 7.4. Brains were then sectioned on a vibratome and sections containing the BLA were placed in ice cold aCSF w/ actinomycin D (93 mM N-methyl-D-glucamine, 2.5 mM KCl, 1.2 mM NaH2FACs sorting for tdTomato positive cells was performed as previously described (Jensen-Cody et al., 2020). Hoechst cell viability dye (Hoechst 33258) was added to the cell suspension at a final dilution of 4ug/ml just prior to sorting to allow for selection of living cells during FACs sorting. Cells were then FACs sorted for tdTomato fluorescence and viability via Hoechst staining. Sorting was performed on a BD FACS Aria II using a 130 mM nozzle, a sheath pressure of 10 p.s.i., and in the single cell sorting mode at the University of Iowa Flow Cytometry Facility.PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 10 mM MgSO4, 0.5 mM CaCl2, 5 mM sodium ascorbate, 2 mM thiourea, and 3 mM sodium pyruvate, 45 µM actinomycin D, pH of 7.3-7.4, bubbled with carbogen gas (95% O2 and 5% CO2) on ice for at least 15 minutes prior to dissection). The BLA was then dissected from each section, and BLA tissue derived from three mice were pooled for each experimental condition. The tissue was then minced and placed in 2mg/ml Pronase in aCSF w/ actinomycin D and incubated at 37°C for 30 minutes. Following digestion, the Pronase solution was exchanged with ice-cold, carbogen-bubbled ACSF containing 1% fetal bovine serum and .01% BSA. The tissue was then titrurated using fire polished Pasteur pipets and strained through a 70 µM cell strainer. FACS isolated cells were then loaded on the BD Rhapsody where capture of the single cells was performed per manufacturer instructions. Following lysis of the cells and capture of mRNA on oligonucleotide barcoded beads the cDNA synthesis was performed and cDNA libraries with Illumina sequencing adapters were generated per manufacturer instructions using the BD Rhapsody whole transcriptome analysis amplification kit. cDNA libraries were then sequenced on Illumina HiSeq 4000 sequencers at the University of Iowa Genomics Core. Sequencing data was then quality filtered and annotated using the BD Rhapsody analysis pipeline while also correcting for artifacts which arise during library construction. Filtered and annotated data from vehicle and FGF21 treated mice were then normalized in the R package Seurat using SCTransform and integrated using Seurat scRNA-seq integration (Stuart et al., 2019). Imputation was performed using the Seurat Wrapper ALRA on the integrated data set before final clustering (clustering resolution was set at 0.5 based on the number of cells sequenced)(Table S2) and gene expression analysis to account for dropout associated with 3'-barcoding-based approaches (Ziegenhain et al., 2017). “Cell type” classification of the clusters was determined based on expression of previously identified marker genes for neuronal and non-neuronal cell types.