GSM5736003: testis - region R2 - 5'end-triphosphate_rep1; Ascaris suum; ncRNA-Seq
Male and female germline tissues were extracted from worms and washed in PBS, frozen whole or following dissection in liquid nitrogen, and stored at -80°C. Argonaute IPs were carried out using affinity purified rabbit antibodies pre-bound to Protein A Dynabeads (Fisher Scientific) (5-10 ug of antibody per 100 ul of beads). Germline or embryo whole cell lysates were mixed with Protein A Dynabeads and rotated overnight at 4°C. Protein A Dynabeads were recovered and washed with high-salt buffer (50 mM Tris–HCl, pH 7.4, 1M NaCl, 1mM EDTA, 1% Igepal CA-630, 0.1% SDS, 0.5% sodium deoxycholate) three times. The beads were resuspended in 250 ul of Proteinase K buffer containing 200 µg/ml Proteinase K and incubated for 1 hr at 37°C. RNA was extracted using Trizol LS (Invitrogen) adapted for small RNA recovery by precipitation using 2 volumes of isopropanol, 15 µg of GlycoBlue (Invitrogen), and 30 minutes incubation at -80°C followed by centrifugation at 18,500 x g for 35 minutes at 4°C. The RNA was treated with RppH (25 units, New England Biolabs) in Thermopol buffer for 5' independent cloning. Both untreated and RppH-treated samples were done for AsALG-1 and AsALG-4 samples. Following RppH treatment, samples were repurified with Trizol LS (Invitrogen) (adopted for small RNAs extraction) and stored at -80°C. RNA was isolated using Trizol or LS Trizol (Invitrogen). Total RNA samples were fractionated into small RNA (< 200 nt) and long RNA (>200nt) using the Monarch RNA cleaner (New England Biolabs). Small RNA libraries were prepared using the Small RNA-Seq Library Prep Kit (Lexogen). Both 5'-phosphate and 5'-phosphate independent libraries (RppH-treated) were prepared. Libraries from 4-cell embryos were also prepared using 18-30 nt gel purified RNA using the SMARTer smRNA-Seq Kit (Clontech) and NEXTflex-Small-RNA-Seq (New England Biolabs) with similar results. RNA >200 nt was treated with TURBO DNase (Ambion) and rRNA depletion carried out using RiboCop rRNA Depletion Kit for Human/Mouse/Rat (Lexogen). Long RNA (>200 nt) libraries were made using CORALL Total RNA-Seq Library Prep Kit (Lexogen). Both small and long RNA libraries were sequenced 150 nt from both ends using the Illumina NovaSeq 6000 System.