GSM6799937: ME13_50um; Mus musculus; RNA-Seq
The fresh frozen tissue section was fixed with formaldehyde. The adapters loaded Tn5/pA-Tn5 transposition containing a ligation linker was inserted into transposase accessible genomic DNA loci. The biotin decorated poly-T primer with a ligation linker was added to capture the mRNA for reverse transcription. In situ ligation was conducted by introducing distinct spatial barcode Ai (i = 1-50/100) to the adapters through an array of lateral microchannels. Then, distinct spatial barcode Bj (j = 1-50/100) were introduced through the longitudinal microchannels. Barcodes A and B with linkers were ligated to the 5' end of the Tn5//pA-Tn5 oligo separately during each ligation. The tissue can be spatially barcoded with a distinct combination of barcodes Ai and Bj (i = 1-50/100, j = 1-50/100, n of barcoded pixels = 2,500/10,000). To correlate the spatially chromatin accessibility, transcriptome, and tissue morphology, the tissue was imaged after each ligation. Reverse crosslinking was then performed to collect barcoded cDNA and DNA fragments. The streptavidin beads were used to separate DNA and cDNA fragments. The cDNA fragments can be enriched with streptavidin beads and the DNA fragment was left in the supernatant. After separation, the DNA and cDNA libraries were constructed separately during PCR amplification. NGS sequencing was then performed using a NovaSeq 6000 sequencer with pair-end 150 bp mode.