GSM6294361: treatment3; Mus musculus; RNA-Seq
1. sn-seq: The lateral ventricles including the choroid plexus and an about 2 mm thin layer of tissue surrounding the ventricles were carefully micro-dissected and homogenized using scissors. The resulting homogenates were filtered through a 40 µm cell strainer and centrifuged at 500 g for 5 min to pellet nuclei. To purify nuclei, the pellet was resuspended and centrifuged at 25,000 rpm at 4 ℃ for 2 hr using an ultra-centrifuge equipped with swinging bucket rotor. The supernatant was carefully removed via aspiration. 1 ml of stain buffer was added to resuspend the nuclear pellet. The nuclei were pelleted at 500 g for 10 min at 4 ℃. Stain buffer with mouse anti-NeuN conjugated to Alexa-Fluor-488 was added. The samples were incubated for 30 min at 4 ℃ and then centrifuged for 5 min at 500 g to pellet nuclei. The pallets were resuspended in stain buffer that contained 0.1 µg/ml DAPI. Single nucleus sorting was carried out on FACSAria III. Nuclei were gated on their morphology and DAPI signal. Nuclei negative for NeuN were sorted. 2. ST: EO771-VM bearing mouse brains were embedded in Tissue-Tek O.C.T. Compound and frozen in isopentane on liquid nitrogen. The embedded mouse brains were cryosectioned coronally to a thickness of 10 mm (at -0.6 mm from the bregma) slice on a cryostat. Sections were placed respectively on Visium Spatial Tissue Optimization Slide and Visium Spatial slide within the capture area. 3. RNA-seq: The different mRNA profiles of HCPEpiC cells treated with or without 500 ng/mL recombinant human tryptase  were compared by RNA-seq. Total RNA was extractedusing the TRIzol  reagent according to the manufacturer's instructions. RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies). 1. sn-seq:Single nucleus was loaded on a 10x GENOMICS GemCode Single-nucleus instrument that generated single-nucleus Gel Bead-In-EMlusion (GEMs). Reverse transcription and libraries preparation were performed using the Chromium Next GEM Single nucleus 3' Reagent Kits v3.1 (10x GENOMICS, CG000204) according to the manufacturer's protocol. Libraries were sequenced on an lllumina NovaSeq 6000 system.  2.ST: After permeabilization, tissues were degraded and removed from the slides. Reverse transcription mix containing SuperScript III reverse transcriptase  was added to each section and incubated overnight at 42 ℃ to generated cDNA with spatial barcode information. The spatially barcoded, full-length cDNA was amplified via PCR to generate sufficient mass for library construction. The libraries were sequenced on the lllumina NovaSeq 6000 system using paired-end sequencing. 28 bases were sequenced on read 1 to determine the spatial barcode and UMIs, and 120 bases were sequenced on read 2 to cover the genetic region. 3. RNA-seq: Libraries of RNA-seq were prepared using NEBNext Ultra RNA Library Prep Kit for Illumina. RNA-seq was performed using the Novaseq6000  with paired-end reads.