title: GSM4656183: hip142078 [Nanopore]; Mus musculus; RNA-Seq
library_construction_protocol: The Visium Spatial Tissue Optimization Slide & Reagent kit (10X Genomics, Pleasanton, CA, USA) was used to optimize permeabilization conditions for mouse brain tissue. Spatially barcoded full-length cDNA was generated using Visium Spatial Gene Expression Slide & Reagent kit (10X Genomics) following the manufacturer's protocol. Tissue permeabilization was performed for 6 and 9 min (hemisphere) and 12 min (MOB). cDNA amplification was conducted with 12 (hemisphere) and 17 (MOB) cycles. 10µl of cDNA library followed 10x Genomics Visium library preparation protocol of fragmentation, adapter ligation, and indexing. The libraries were sequenced on a NextSeq500 (Illumina), with 28 bases from read 1 and 91 from read 2. Nanopore sequencing libraries were prepared with the LSK-109 kit from Oxford Nanopore (1 µg cDNA) following the instructions from the manufacturer. PromethION flowcells were loaded with 200 ng library each. PCR amplifications for Nanopore library preparations were made with Kapa Hifi Hotstart polymerase (Roche Sequencing Solutions): initial denaturation, 3 min at 95°C; cycles: 98°C for 30 sec, 64°C for 30 sec, 72°C for 5 min; final elongation: 72°C for 10 min, primer concentration was 1 µM.