Quick cip

RNAs were pretreated to modify 5’ ends in two ways. For RPPH treatment, 1-2 μg of total RNA was treated with 10 units (2 μl) RNA 5’ pyrophosphohydrolase (NEB) for 1 h at 37°C. The treated RNA was phenol-chloroform extracted and ethanol precipitated with sodium acetate and glycogen for 2 days, and resuspended in RNase-free water. For CIP-RPPH treatment, 3-5 μg of total RNA was treated with 4 μl of QuickCIP (Quick dephosphorylation kit, NEB) in a total volume of 40 μl for 90-120 min at 37°C. RNA was phenol-chloroform extracted, precipitated overnight with sodium acetate and glycogen and resuspended in RNase-free water.
Small RNA libraries from treated or untreated RNA were built using the TruSeq small RNA kit (Illumina) according to the manufacturer’s instructions except for an increase in the number of PCR cycles from 11 to 15. Libraries were eluted in 0.3 M NaCl, ethanol precipitated and quantitated with Qubit and TapeStation. Libraries were pooled in groups of 6 to 12 per lane and sequenced on an Illumina HiSeq2000.
The Illumina universal adapter was trimmed from small RNA reads using cutadapt v1.10 and reads were mapped to the corresponding genome assemblies with Bowtie v0.12 (Langmead et al., 2009 (link)) with parameters –v 0 –m 1.

GGPP was synthesized as previously described in the literature^{63 (link),64 (link)}. In vitro reactions were performed in 50 mM Tris pH 6.8, containing 1 mM MgCl₂, 5 mM β-mercaptoethanol (BME), 10% glycerol, 4 mM GGPP, and 20 µM Tpn2 in a total volume of 50 µL. Reactions were incubated for 16 h at 30 °C before undergoing dephosphorylation of reaction products via addition of Quick-CIP (NEB) and incubation following the manufacturer’s protocols. The reaction mixture was then gently extracted with 100 µL acetonitrile and 50 µL saturated NaCl before centrifugation for 1 min. The organic layer was then removed and injected on an HPLC system using an isocratic method of 95% acetonitrile in H₂O. The substrate and product were detected at 210 nm as the dephosphorylated analogs 1′ and 2′ and eluted at 5.4 min and 6.0 min, respectively.
TPP (2) was isolated by scaling up in vitro reactions to yield approximately 0.5 mg of the product. Reactions were incubated for 16 h at 30 °C before extraction with equal volume of acetonitrile and placing on ice for 1 h. TPP (2) was purified via preparative HPLC using a solvent gradient of 5–50% acetonitrile in NH₄HCO₃. Fractions containing 2 were combined and dried down via rotary evaporator (Supplementary Figs. 3, 4).

100 ovaries per sample were used in Aub and Piwi IP experiments and processed, as previously described (46 (link)). For Ago3 IP experiments, we used 150 ovaries per sample with a slight modification in the protocol. Ovary lysates were first incubated with 4 μg Ago3-380 antibody for 2 h at 4°C and then mixed with buffer-equilibrated Protein G Dynabeads (Thermo Fisher Scientific) for 90 min at 4°C. The RNAs associated with immunopurified PIWI proteins were extracted with TRIzol reagent (Ambion) and dephosphorylated with Quick CIP (NEB) in CutSmart buffer for 10 min at 37°C. After enzyme inactivation for 2 min at 80°C, a T4 PNK (NEB) labeling reaction was set in 1× CutSmart buffer with the addition of DTT to 5 mM in the presence of ^γ32PATP. Reactions were run with 8 M urea 15% PAGE.

Design and cloning details for all genetic components utilized in this study are provided in Supplementary Tables S1 and S2 (Supporting Information). Plasmids were designed using Benchling (www.benchling.com) and constructed by standard molecular cloning techniques. For restriction enzyme-based cloning, plasmids were digested with the desired endonucleases (New England Biolabs); digested backbones were dephosphorylated with Quick CIP (M0525L, New England Biolabs) before ligation with T4 DNA ligase (EL0011, Thermo Fisher). All PCR reactions were performed using Q5 High-Fidelity DNA polymerase (M0491L, New England Biolabs). After ligation, the plasmids were transformed and amplified in E. coli XL10-Gold strain (Agilent) and DNA was extracted using a plasmid miniprep kit (Zymo Research) or Midiprep Kit (D4200, Zymo Research). Constructs were verified by Sanger sequencing at Microsynth AG. Synthetic gene fragments used in the study were codon-optimized for expression in human cells and synthesized by Twist Bioscience.

For the initial analysis of NTD RNA-binding preferences, radioactive EMSAs were performed according to reference^{88 (link)} with the following modifications: 30 pmol RNA transcripts were dephosphorylated using Quick CIP (NEB) following the manufacturer’s protocol and resuspended in H₂O. Subsequently, 5′ end-labeling of 15 pmol RNA transcripts with [γ-³²P]-ATP was accomplished with T4 polynucleotide kinase (NEB). Labeled RNA was separated from unincorporated [γ-³²P]-ATP by column purification (NucAway) and adjusted with NTD buffer (25 mM potassium phosphate, 150 mM potassium chloride, pH 6.5) to 0.03 pmol/μl. Binding was performed for 10 min at RT in 20 μl reaction volume in the presence of 0.6 μg tRNA from baker’s yeast (Sigma), 3 nM ³²P-labeled RNA, 1 mM MgCl₂ and various dilutions of NTD in NTD buffer. After the addition of 3 μl loading buffer (30% glycerol, bromphenol blue, xylene cyanol) the RNP complexes were resolved by PAGE (6% polyacrylamide, 5% glycerol, and 1×TBE) at 80 V for 75 min at 23 °C, with pre-cooled (4 °C) TB running buffer (0.13 M Tris, 45 mM boric acid) for improved resolution. Gels were dried and subsequently exposed to a phosphor imager screen and visualized using a Typhoon laser scanner (GE). Images were exported using ImageQuant TL (v8.1).

DNA fragments were PCR amplified from plasmids in 800 μl volumes using a Q5 High-Fidelity PCR kit (NEB) following the manufacturer’s recommended procedure. After amplification, the reaction was then passed through a single column from a Monarch PCR & DNA cleanup kit (NEB) following the manufacturer’s recommendations for double-stranded DNA <2 kb. The column was eluted with 15 μl elution buffer and digested with 10 U AvaI at 37 °C for 2 h. For KLHL15 promoter fragments only, before digesting with AvaI, the eluate was digested with 10 U SmaI at 37 °C for 1 h followed by treatment with 5 U QuickCIP (NEB) for 30 min at 37 °C and heat inactivation for 10 min at 80 °C. The reactions were stopped by adding 4 μl 6× Purple Gel Loading Dye (NEB) and then run on a 0.8% Tris:acetate:EDTA agarose gel. Fragments were gel purified using a QIAquick gel extraction kit (Qiagen) according to the manufacturer’s recommendations and were further concentrated by ethanol precipitation.