Name: NvElav::mOrange pos R2_s
Instrument: NextSeq 500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: Oligo-dT
Layout: SINGLE
Construction protocol: Animals were dissociated as described in Torres-Mendez et al., Nat Ecol Evol 2019. Cell suspensions were sorted by FACS to separate NvElav1::mOrange -positive from -negative cells. Total RNA was extracted with the Direct-zol RNA MicroPrep kit (Zymo Research). cDNA was prepared from 400pg of total RNA using the Smart-Seq 2 method with 16 pre-amplification PCR cycles, as described by Picelli et al. (2014). NGS libraries were prepared using the home-made tagmentation-based method as described by Hennig et al., 2018. Briefly, 125 ng of cDNA was tagmented using home-made Tn5 loaded with annealed linker oligonucleotides for 3 minutes at 55C. Reaction was inactivated by adding 1.25ul of 0.2% SDS and incubation for 5 minutes at room temperature. Indexing and amplification was done using the KAPA HiFi HotStart PCR kit (Sigma-Aldrich) with Index oligonucleotides (sequences were adapted from Illumina).