BioSample

Protocols: We performed an NvLsd1GFP/- x NvLsd1GFP/- cross. 75% of the animals have at least one copy of the NvLsd1GFP allele, hereafter called control, and the homozygous mutant animals are GFP negative, hereafter NvLsd-/-. Anaimals were separated based on whether they were GFP negative or positive and 50 animals were used per sample. 50 animals per sample were lysed in 500 μl TRIzol reagent (Invitrogen, 15596026) by vortexing extensively and incubated at RT for 5 minutes. 100 μl of chloroform was added and mixed vigorously and the aqueous component was isolated using MaXtract High Density tubes (Qiagen, 129046) using the manufacturers protocol. One volume of 100% Ethanol was added to the aqueous phase. This was then processed using an RNeasy Micro Kit (Qiagen, 74004) using the manufacturers protocol including on column DNase digestion using the RNase-Free DNase Set (Qiagen, 79254). RNA quality was assessed using an RNA 6000 Pico Kit (Aligent, 5067-1513) and concentration determined using the Qubit™ RNA HS assay kit (Invitrogen, Q32852). Libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (E7760L), with following changes: 25ng RNA input, 1/100 adaptor dilution, 14 PCR cycles.