title: NextSeq 500 sequencing; RNA-seq of NvElav1::mOrange positive and NvElav1::mOrange negative cells from Nematostella vectensis
design: RNA-seq of NvElav1::mOrange positive and NvElav1::mOrange negative cells from Nematostella vectensis
library_name: NvElav::mOrange pos R4_s
library_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).
attributes.tag: Experimental Factor: phenotype
attributes.value: NvElav1::mOrange positive