title: GSM3184866: CTRL_small_4; Drosophila melanogaster; ncRNA-Seq
library_construction_protocol: RNA was extracted from Drosophila heads using a standard Trizol protocol. Library preparation and sequencing were performed by the Genome Sequencing Facility at Greehey Children's Cancer Research Institute at the University of Texas Health San Antonio. For standard RNA-seq, three independent biological replicates were sequenced, each consisting of 500 ng of total RNA from six pooled Drosophila heads (18 heads total). Extracted RNA was used for library preparation according to the KAPA Stranded RNA-seq Kit with RiboErase (HMR) sample preparation guide.  For small RNA-seq, four independent biological replicates were sequenced. 500 ng of total RNA from pooled Drosophila heads was used for library preparation according to the NEBNext small RNA sample preparation guide. Due to the abundance of the 2S rRNA in Drosophila, we included an additional 2S block step using the oligo (5'-TAC AAC CCT CAA CCA TAT GTA GTC CAA GCA/3SpC3/-3') as described48. 2S rRNA blocking was performed directly after 3' SR adapter ligation. 1 μM of the 2S rRNA block oligo was added directly to each ligation reaction on ice, and reactions were incubated at 90°C for 30 sec, then 65°C for 5 min. After 5 min, 1 μl of SR RT primer was added and we proceeded as described in the NEBNext protocol.