title: FgDicer2_mutant_RNA-seq_sample_3
design: Approximately 0.3 g of frozen powdered sample was resuspended in 1.5 ml of polysome extraction buffer (PEB)/lysis buffer [50 mM Tris-HCl (pH 8), 0.2 M Sucrose, 0.2 M KCl, 15 mM MgCl2, 2% (v/v) Polyoxyethylene (10) tridecyl ether (Sigma P2393), 1% triton X-100, 100 g/mL chloramphenicol, 20 mM 2-Mercaptoethanol, 100 g/mL cycloheximide, and 10 unit/mL DNase I] and incubated on ice for 10 min with gentle shaking. The lysate was centrifuged at 15,000  g for 12 min and the supernatant was transferred to new tubes by dividing into 1 ml and 0.5 ml aliquots, flash frozen and stored at -80C. For footprint purification, 1 ml of stored lysate was divided into 0.5 ml aliquots and 6.5 L of RNase I (100U/L) was added to each tube (Thermo Fisher Scientific; AM2294). The digestion was carried out at room temperature for 1 h with gentle rotation and stopped by adding 15 L SUPERaseIn RNase Inhibitor (Thermo Fisher Scientific; AM2696). The digestion mixture was layered onto 0.4 ml of sucrose cushion (containing 40 mM Tris-HCl (pH 8), 1 M sucrose, 0.1 M KCl, 15 mM MgCl2, 100 g/mL chloramphenicol, 10 mM 2-Mercaptoethanol, 100 g/mL cycloheximide, 20 U/ml SUPERaseIn) in thickwall 11 x 34 mm polycarbonate tubes (Beckman Coulter Life Sciences; 343778).  Samples were weighed and balanced by adding PEB and centrifuged in TLA100.3 rotor at 100,000 rpm for 1.4 h in a pre-chilled ultracentrifuge (Beckman coulter Optima MAX-XP Ultracentrifuge). The supernatant was decanted, and the pellet washed with nuclease free water and resuspended in 0.2 ml nuclease free water containing 10% SDS.  RNA was extracted from the resuspended pellet by adding 1 ml Triazol reagent (Invitrogen), vortexed and kept rotating in room temperature for 10 min and 0.2 ml chloroform was added, vortexed and centrifuged at 13,000 g for 20 min at room temperature. The aqueous upper phase was transferred to a new tube and 0.7 ml isopropanol was added to precipitate the RNA for overnight at -20C. Tubes were centrifuged at 15,000 g for 45 min at 4 C to pellet the RNA. The pellet was resuspended in 7 L of 10 mM Tris buffer (pH 8) and stored at -80C. For RNA isolation, 1 ml Trizol was added to 0.5 ml stored lysate, and RNA was purified as explained above with final resuspension in 20 L 10 mM Tris buffer (pH 8) and stored at -80 C. RNA samples were treated with DNase I (1 U/L, Thermo Fisher Scientific; EN0521) by incubating at 37C for 30 min and ethanol precipitation was performed. Approximately 8-10 g of DNA free total RNA (20 L) was randomly fragmented with equal an volume of 2 alkaline fragmentation solution (2 mM EDTA, 10 mM Na2CO3, 90 mM NaHCO3, pH  9.3) and the reaction was incubated at 95 C for 25 min. Fragmentation was stopped by the addition of 0.56 ml ice-cold precipitation solution (300 mM NaOAc pH 5.5, plus GlycoBlue (Ambion) as a co-precipitant), followed by the addition of 0.6 ml of isopropanol and precipitated on ice for 1 h RNA was then pelleted by centrifugation for 30 min at 20,000 g, 4C in a tabletop centrifuge. The supernatant was removed by pipetting and the pellet air-dried for 10 min before resuspension in 15 L of 10 mM Tris pH 8. The fragmented RNA was subjected to T4 polynucleotide kinase (T4 PNK) treatment to 3` end dephosphorylation and 5` end phosphorylation as stated above. RNA was purified using RNA Clean & Concentrator kit (Zymo Research; R101750) followed by phenol-chloroform method detailed in the kit protocol. NEBs NEBNext Multiplex Small RNA Library Prep Set was used for preparing small RNA libraries and libraries were normalized to 2 nM and pooled for sequencing on the Illumina NovaSeq 6000 with 100 bp single reads.