The spatially barcoded tissue was enclosed with a clean polydimethylsiloxane (PDMS) reservoir and securely clamped using acrylic chips. A 2X lysis buffer was prepared in advance, consisting of 20 mM Tris-HCl pH 8.0, 400 mM NaCl, 100 mM EDTA, and 4.4% SDS. For tissue digestion, 70 µL of the lysis mix (30 µL 1X DPBS, 30 µL 2X lysis buffer, 10 µL 20 µg/µL Proteinase K solution) was loaded into the PDMS reservoir, sealed with parafilm, and incubated in a humidified box at 55°C for 2 hours. After the reaction, the parafilm was removed, and all the liquid containing cDNA was collected into a 1.5mL DNA low-bind tube. PCR amplification was executed to directly ligate sequencing primers, using a 100 µL system consisting of 50 µL 2X KAPA HiFi HotStart ReadyMix, ~42 µL solution from the rRNA removal step, 4 µL 10 µM P5 Primer, and 4 µL 10 µM P7 Primer. The resulting library underwent purification using SPRIselect beads at a 0.8X ratio, quality control checked using TapeStation, and was then sequenced on an Illumina NovaSeq 6000 Sequencing System with a paired-end 150bp read length.