title: GSM6722696: Telencephalon, not building, biol rep 1, capture area B; Mchenga conophoros; RNA-Seq
library_construction_protocol: Telencephalon tissue samples were embedded in cryomolds (7mm x 7mm x 7mm) containing chilled OCT (Optimal Cutting Temperature embedding compound) and snap frozen. OCT-embedded tissue blocks were cryosectioned at 10 m thickness at -20C (CryoStar NX70). Tissue sections were mounted onto 6.5 mm2 capture areas on Visium Spatial Gene Expression Slides (10X Genomics). There are four capture areas per slide, each containing 5000 spatially-barcoded 55m-diameter spots. RNA quality of the tissue sections (RIN > 7) was confirmed using the Agilent RNA 6000 Nano Kit on the Bioanalyzer 2100 system (Agilent). Gene Expression slides containing tissue sections were processed according to manufacturer instructions (Methanol Fixation, H&E Staining, and Imaging – Visium Spatial Protocol (10X Genomics)). Slides were fixed in methanol for 30 minutes at -20C and stained with Hematoxylin and Eosin (H&E). Brightfield images of H&E-stained slides were taken at 1.33 m/pixel resolution (5X objective, Zeiss AxioObserver Z1 Fluorescent Microscope) and stitched using Zen 2 software (blue edition, Zeiss) prior to library construction. Dual-index Illumina paired-end spatial gene expression libraries were prepared according to manufacturer instructions (Visium Spatial Gene Expression Reagent Kits User Guide (10X Genomics)). Fixed and stained tissue sections were permeabilized for 18 minutes and poly-adenylated mRNA released from cells was captured by primers on the underlying gene expression spots. Primers include a 10X spatial barcode, unique molecular identifier (UMI) and an Illumina read 1 sequencing primer. Incubation with reverse transcription (RT) reagents produced spatially barcoded, full-length cDNA. Barcoded cDNA was denatured, transferred into tubes, and amplified via PCR. Amplified cDNA was size-selected with SPRIselect. During final library construction, P5 and P7 paired-end construct sequences, i5 and i7 sample indices, and an Illumina read 2 primer sequence were added. Quality was assessed using high sensitivity DNA analysis on the Bioanalyzer 2100 system (Agilent).