GSM9438232: MouseEmbryo_E11_50um_pixel_adjacent_mRNA; Mus musculus; OTHER
The barcoded tissue ROI was enclosed with a clean PDMS reservoir and clamped using acrylic chips. A 2X lysis buffer (20 mM Tris-HCl pH 8.0, 400 mM NaCl, 100 mM EDTA, 4.4% SDS) was prepared. For tissue digestion, 70 μL of lysis mix (30 μL 1X DPBS, 30 μL 2X lysis buffer, 10 μL Proteinase K at 20 μg/μL) was added to the PDMS reservoir, sealed with parafilm, and incubated at 55°C for 2 h. After incubation, the liquid containing cDNA was collected, and 40 μL of fresh lysis mix was added to recover any remaining cDNA. The tissue lysate was incubated overnight at 55°C to reverse crosslinks. Nucleic acids were then extracted by phenol-chloroform (pH 8.0), ethanol-precipitated, and dissolved in 20 μL of water. Biotinylated cDNA was enriched with streptavidin beads. A 3' cDNA adapter was ligated using T4 RNA ligase 1, with incubation at 25°C for 16 h. The cDNA was subsequently recovered from the beads. Libraries were prepared via PCR amplification using NGS sequencing primers. The amplified libraries were size-selected to a range of 280–500 bp using a 6% Novex TBE gel.