GSM1544078: XL_Xiwi_RIPseq; Xenopus laevis; RIP-Seq
Whole cell lysates for CLIP were generated by lysing in Q-column buffer (20mM Hepes-KOH pH 7.9; 10% glycerol; 0.1 M KOAc; 0.2 mM EDTA; 1.5 mM MgCl2; 0.5 mM DTT; 1X Roche Complete EDTA-free Protease Inhibitor Cocktail; 0.5% NP40), dounced with 50 strokes and sonicated 2 rounds for 30 secs.  mRNA was CLIP RNA fragments were extracted with Trizol. mRNAs were further isolated by the Magnetrac Atail selection kit. The two antibodies used are not sold from a manufacturer but were custom made.  The mouse monoclonal antibody against Piwi was obtained from the Siomi lab (Saito et al. Genes Dev. 2006 Aug 15;20(16):2214-22.) while the rabbit polyclonal antibody against Piwi was generated by the Lau lab with a peptide antigen to the N terminal 12 amino acids of the Piwi gene. For  RIPseq, sRNAseq and oocyte RNAseq experiments, whole cell lysates were generated from stage 1-3 oocytes only. Both ovaries were dissected from an adult frog and the oocytes were liberated from the follicle cells by cutting the ovaries into ~3mm2 peices and incubating them in 3mg/mL collagenase in 0.1M Sodium Phosphate pH 7.4 for ~1hour. Liberated oocytes were then washed into Modified Barth's Saline (5mM HEPES-KOH pH 7.8, 88mM NaCl, 1mM KCl, 1mM MgSO4, 2.5mM NaHCO3, 0.7mM CaCl2) and passed through a 450µm mesh screen to separate out the stage 1-3 oocytes. Stage 1-3 oocytes were washed into XB buffer (10mM HEPES-NaOH pH7.7, 1mM MgCl2, 0.1mM CaCl2, 100mM KCl, 50mM Sucrose, 1x Protease inhibitor mix) and Dounced with 2 strokes. Lysed oocytes were spun at 20,000xg for 5 minutes at 4˚C. The middle layer containing cytosol and organelles was saved as the stage 1-3 oocyte extract. CLIP tags and one set of mRNA sequencing libraries were generated with custom protocol for constructing small RNA libraries (Robine et al, Curr Bio 2009).  All other mRNA libraries were constructed with the ScriptSeq version 1 and version 2 kits from Epicenter Biotech. RIPseq and XL_Oocyte_RNAseq libraries were generated using the Illumina Tru-Seq RNA kit. Xiwi and Xili sRNAseq libraries were constructed using a linker ligation protocol adapted from a custom protocol (Ingolia et al, Nature Protocols 2012).