title: GSM9239728: FF_Mouse_Embryo_RNaseH_V2; Mus musculus; OTHER
library_construction_protocol: Fresh frozen (FF) samples were OCT embedded. Both FF and FFPE samples were sectioned at a thickness of 7-10 μm and mounted on the center of poly-l-lysine-covered glass slides (63478-AS, Electron Microscopy Sciences). E11 mouse whole embryo paraffin sagittal sections were made of freshly harvested tissues, fixed in 10% neutral buffered formalin, and processed for paraffin embedding. Paraffin blocks were also sectioned at a thickness of 7-10 μm and mounted on the center of poly-l-lysine-covered glass slides. De-identified archived formalin-fixed paraffin-embedded (FFPE) human reactive lymph node and lymphoma tissue blocks were obtained from Yale Pathology Tissue Services (YPTS). The tissue retrieval and distribution for research was conducted with the approval of the Yale University Institutional Review Board (Approved IRB #1401013259) and oversight by the Tissue Resource Oversight Committee. Written informed consent for participation in any cases where identification was collected alongside the specimen, was obtained from patients or their guardians, in accordance with the principles of the Declaration of Helsinki. Each sample was handled in strict compliance with HIPAA regulations, University Research Policies, Pathology Department diagnostic requirements, and Hospital by-laws. For human samples, after review of all slides by board-certified pathologist and selection of the optimal block, paraffin blocks were sectioned at a thickness of 7-10 μm and mounted on the center of Poly-L-Lysine coated 1 x 3" glass slides. The sectioning was carried out by YPTS. Paraffin sections were stored at −80°C until use. The SEQuoia RiboDepletion Kit was used to remove rRNA and mitochondrial rRNA from the amplified cDNA, following the manufacturer's instructions. 20 ng of cDNA was used as the input amount, and three rounds of depletion were performed. We did observe that two rounds of depletion could suffice. Next, following the PCR steps described in the previous step, 10 PCR cycles was sufficient 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 Modified N501 Primer, and 4 μl 10 μM P7 Primer. The library product was purified using SPRIselect beads at a 0.8X ratio, according to the manufacturer's standard protocol.