title: GSM5513606: Spatial_transcriptomics_5 weeks (CS15) human embryo_4; Homo sapiens; OTHER
library_construction_protocol: CS15d embryo in toto was frozen in OCT medium and stored at −80°C until sectioning. Optimization of tissue permeabilization was performed on 10-μm-thick sections using Visium Spatial Tissue Optimization Reagents Kit (10X Genomics, Pleasanton, CA, USA), which established an optimal permeabilization time of 30 minutes. Samples were mounted onto a Gene Expression slide (10X Genomics), fixed in ice-cold methanol, stained with hematoxylin and eosin, and scanned with Aperio scanner (Leica Biosystems, Germany). Tissue permeabilization was performed to release the poly-A mRNA for capture by the poly(dT) primers precoated on the slide, which include an Illumina TruSeq Read, spatial barcode, and unique molecular identifier (UMI). Visium Spatial Gene Expression Reagent Kit (10X Genomics) was used for reverse transcription to produce spatially barcoded full-length cDNA and for second strand synthesis followed by denaturation to allow a transfer of the cDNA from the slide into a tube for amplification and library construction. Visium Spatial Single Cell 3' Gene Expression libraries consisting of Illumina paired-end sequences flanked with P5/P7 were constructed after enzymatic fragmentation, size selection, end repair, A-tailing, adaptor ligation, and PCR. Dual Index Kit TT Set A (10X Genomics) was used to add unique i7 and i5 sample indexes and generate TruSeq Read 1 for sequencing the spatial barcode and UMI and TruSeq Read 2 for sequencing the cDNA insert, respectively. Sequencing was performed on Illumina NovaSeq 6000.