title: GSM8568361: Hepatoblastoma, LCM-9, Embryonal; Homo sapiens; RNA-Seq
library_construction_protocol: Fresh sections of formalin-fixed paraffin-embedded tumor blocks were cut on a microtome at 7 micron thickness and mounted on glass slides with polyethylene naphthalate membranes and stored in a nitrogen chamber. In preparation for laser capture microdissection, slides were immersed sequentially for 20 seconds each in xylenes (three times), 100% ethanol (three times), 95% ethanol (two times), 70% ethanol (two times), water, hematoxylin, water, bluing reagent, water, 70% ethanol (two times), 95% ethanol (two times), 100% ethanol (three times), and xylenes (three times). Immediately after staining, cells were dissected on an ArcturusXT LCM System using the ultraviolet (UV) laser to cut the sample and the infrared laser to adhere it to a CapSure HS LCM Cap. Roughly 500 cells were captured by area, based on density estimates by cell counting on small areas. After LCM, the cap was sealed in a 0.5 mL tube and stored at −80°C until library preparation. The Smart-3SEQ protocol is available here: https://genome.cshlp.org/content/suppl/2019/10/24/gr.234807.118.DC1/Supplemental_File_2.pdf. Sequencing libraries were prepared using the Smart-3SEQ protocol for FFPE tissue on an Arcturus LCM HS cap, with the pre-SPRI pooling option and a single batch for all samples. Libraries were characterized immediately and stored at −20°C until sequencing. Sequencing was performed on the pooled library using the NextSeq 500/550 High Output V2 kit with 75 cycles (read 1: 76 cycles, index 1: 2bp, index 2: 8bp)