Visium spatial library construction kit

Mouse kidneys were snap-frozen in liquid nitrogen and embedded in OCT (Tissue-Tek) for preparation of 10-μm cryosections. For tissue visualization, frozen tissue sections were fixed with pre-chilled methanol on the Visium Tissue Optimization Slides (10X Genomics, PN-1000193), followed by staining with hematoxylin (S3309, Dako) and eosin (CS701, Dako). Brightfield histological images were taken with a Leica DMI8 whole-slide scanner. For RNA isolation and reverse transcription, sections were permeabilized with permeabilization enzymes to release mRNA, which was captured by probes on the Visium spatial gene expression slides (PN-1000184,10X Genomics). The captured mRNA was reversely transcribed to cDNA, spatially barcoded, amplified, and subjected to library construction using the Visium Spatial Library Construction Kit (PN-1000184,10X Genomics). Briefly, 10 μl of amplified cDNA from each sample was taken for library preparation through the processes of fragmentation, adapter ligation, PCR, and purification. The constructed library was sequenced with an Illumina Novaseq6000 sequencer with a sequencing depth of at least 100,000 reads per spot (performed by CapitalBio Technology, Beijing).

Removing RT Master Mix from the wells after the end of first-strand synthesis. Wells were incubated for 5 min at room temperature after adding 75 μL 0.08 M KOH, then washed with 100 μL EB buffer. Second-strand synthesis was performed by adding 75 μL Second Strand Mix to each well.
Visium spatial libraries were constructed using Visium spatial Library construction kit (10× Genomics, PN-1000184) according to the manufacturer’s instructions. The libraries were sequenced on Illumina Novaseq 6000 platform with pair-end 150 bp (PE150) strategy (performed by CapitalBio Technology, Beijing).