LA_Hydra_Promastigote_Rep4
To perform HydraPsiSeq, total RNA (5ug) was treated with 50% hydrazine (Sigma) for 45 minutes on ice and ethanol precipitated. The RNA pellet was then resuspended in 1M aniline (Sigma) (pH 4.5 adjusted using glacial acetic acid) until the white pellet was completely dissolved, boiled for 15 minutes at 60oC in the dark, and immediately placed on ice. The fragmented RNA was recovered by ethanol precipitation and used for library preparation. To prepare RNA-seq libraries, the fragmented RNA (~800ng) was dephosphorylated with FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific), cleaned by Agencourt RNA clean XP beads (Beckman Coulter) and ligated to a 3 linker using high concentration T4 RNA Ligase 1 (NEB) in a buffer containing DMSO, ATP, PEG 8000, and RNase inhibitor (NEB). The ligated RNA was cleaned from excess linker using Dynabeads MyOne SILANE beads (Thermo Scientific), and first strand cDNA was prepared using the AffinityScript Reverse Transcriptase enzyme (Agilent). The RNA was subsequently degraded using 2ul of 1M NaOH, and the cDNA was cleaned using Dynabeads MyOne SILANE beads (Thermo Scientific). The cDNA was further ligated to a 3 adapter using high concentration T4 RNA Ligase 1 (NEB) and cleaned of excess adapter by using Dynabeads MyOne SILANE beads (Thermo Scientific). The adapter ligated cDNA was PCR enriched using NEBNext High-Fidelity (NEB) polymerase (9 PCR cycles), separated on an E-Gel EX agarose gel (Invitrogen) and size selected in the range of 150-300 bp (containing ~30-180 nt corresponding to RNA). The amplicons were gel purified using NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel), and sequenced in a Nextseq system (Illumina) in paired end mode (20 million reads for each sample).
LA_Hydra_Promastigote_Rep4