GSM8353848: STSW3_S3_L002; Homo sapiens; OTHER
1-2 curls (10um thickness each) were used for RNA extraction using Qiagen RNeasy FFPE kit (Qiagen 73504) according to manufactures' protocol. Extracted RNA was examined by Agilent Bioanalyzer RNA pico chip to confirm the DV200 >30%.  For each FFPE sample, 1 section (5um thickness) was placed on Leica Bond plus slides. Each slide was incubated at 42°C for 3 hours followed by overnight room temperature incubation. Slides were stored in desiccated slide holders until proceeding to deparaffinization. The deparaffinization, HE stains and imaging and decrosslinking of tissue slides were performed according to 10x Genomics protocol (CG000520 Rev B) specific for Visium CytAssist spatial gene expression for FFPE kit. he deparaffinization, HE stains and imaging and decrosslinking of tissue slides were performed according to 10x Genomics protocol (CG000520 Rev B) specific for Visium CytAssist spatial gene expression for FFPE kit. Then, the slides were proceeded to human probe (v2) hybridization and ligation using 10x Genomics Visium CytAssist spatial gene expression FFPE, human transcriptome, 6.5mm kit (10x Genomics, PN-1000520). The probes were released from tissue slide and transferred to barcoded visium slide using Visium CytAssist instrument followed by probe extension. Sequencing libraries were prepared according to manufacturer's protocol. Multiplexed libraries were pooled and sequenced on Novaseq6000 S2 flowcell 100 cycles kit with following parameter: 28nt for Read 1 and 90nt for Read 2. The visium slide processing, library preparation and sequencing was done at Northwestern University NUseq facility core.