title: GSM7350146: heart sample, control patient 7; Homo sapiens; RNA-Seq
library_construction_protocol: Human heart paraffin sections were used to retrieve the PFA-cross-linked mRNA as previously described (reference below). In brief, PFA-fixed paraffin-embedded hearth tissue sections were scratched from each slide using a stereomicroscope (Olympus SZ51, Model# 1111260100), collected in cold PKD buffer supplemented with proteinase K solution and snap-frozen in liquid nitrogen until further use. To prepare the Smartseq2 libraries, samples were thawed at RT for 3 min and incubated at 56°C in a thermal cycler (lid temperature 66°C), for 4 h or until tissue was completely dissolved. Samples were then placed on ice and incubated at 56°C for 1 min with dT25 magnetic beads to reverse crosslinked samples. Samples were incubated at RT to allow mRNA hybridization and washed with 1x hybridization buffer (HB), containing 2x SSPE, 0.05% Tween-20 and 0.05% RNase Inhibitor. Samples were then washed with PBS with 0.1% RNase Inhibitor and incubated with RNase-free water for 2 min at 80°C to elute mRNA. Smartseq2 libraries were prepared from 1 ng mRNA, as previously described (reference below).  (Reference: Ji, H., Besson-Girard, S., Androvic, P., Bulut, B., Liu, L., Wang, Y., and Gokce, O. (2023). High-Resolution RNA Sequencing from PFA-Fixed Microscopy Sections. In Neural Repair Methods in Molecular Biology., V. T. Karamyan and A. M. Stowe, eds. (Springer US), pp. 205–212. 10.1007/978-1-0716-2926-0_16) Smart-seq2 libraries