5XP-seq method; Pool_B; Replicate_2; D. melanogaster whole embryo
5XP-seq: RNA was isolated using the miRNeasy Micro kit (Qiagen) according to manufacturers instructions. Most reagents are available in NEBNext Multiplex Small RNA Library prep kit for Illumina (New England Biolabs; NEB-kit). The 3SR Adapter for Illumina (NEB-kit) was ligated to 30 ng of input RNA, and a 2S rRNA blocking oligo was added simultaneous to hybridization of the reverse transcription primer in all methods. In 5XP-seq, 5-tagging was then performed by ligating a short RNA oligo. In 5P-seq, the 5 SR Adaptor was ligated instead, and in 5X-seq no 5 ligation was performed on the RNA. Synthesis of cDNA was done using reagents from the NEB-kit. In 5XP-seq and 5X-seq, the second adaptor was ligated to the corresponding 3-end of cDNA using a pre-adenylated R1R adapter mixed with a 2S RNA blocking LNA oligo (custom). No R1R adaptor was ligated in the 5P-seq method that already contained both adaptors. Clean cDNA samples was amplified using either using Phusion High-Fidelity polymerase (ThermoFisher; 5XP-seq and 5X-seq) or Long Amp Taq (NEB-kit; 5P-seq), using the Illumina Multiplex Primer and barcodes (NEB-kit). Cleaned amplified libraries were size selected between 130-200 nt on 6% polyacrylamide gels. Gel extraction was done using Gel breaker tubes (IST Engineering Inc) and released libraries were then ethanol precipitated overnight at -80oC. Library concentrations were estimated using Quantus fluorometer (Promega). Pooled libraries were sequenced on NextSeq 500 with NextSeq 500/550 High Output Kit v2.5, 75 cycles (Illumina).