DNBSEQ-G400 paired end sequencing; Allele-resolved scRNA-seq of primary mouse fibroblasts using Smart-seq3
Allele-resolved scRNA-seq of primary mouse fibroblasts using Smart-seq3
Mouse primary fibroblasts were derived from adult CAST/EiJ x C57BL/6J or C57BL/6J x CAST/EiJ mice (approval by the Swedish Board of Agriculture, Jordbruksverket: N343/12) by skinning, mincing and culturing tail explants of a male adult mouse (12-14 weeks). Cells were lysed in 3 ul of Smart-seq3 lysis buffer and lysate used for library preparation without nucleic acid extraction. Smart-seq3 library preparations were performed as published: https://www.protocols.io/view/smart-seq3-protocol-bcq4ivyw  Library preparation used the following parameters: 1) 20 cycles of PCR for pre-amplification of cDNA, 2) a ratio of 0.6:1 for bead:sample purification of pre-amplified cDNA (using homemade 22% PEG beads), 3) tagmentation of 150 ng bead purified cDNA using 0.1 Î¼L of ATM and 4) 12 cycles of PCR for library amplification of the tagmented samples using custom-designed Nextera index primers containing 10-bp indexes.