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Supp Material.Pdf Supplemental Information SUPPLEMENTAL MATERIALS AND METHODS Vector construction For the construction of pEF4-3xFlag A/B/C vectors, DNA fragment of 3xFlag-tag sequence cloned from pRH3xFlag vector (Saito et al. 2005) was inserted into KpnI and BamHI site of pEF4-Myc/His A/B/C vectors (Life Technologies, V942-20). The FLAG-MILI expression vector was produced by insertion of the Mili cDNAs, amplified form C57BL/6J line mouse testis. Primer sequences are described in Supplemental Table S10. The FLAG-MIWI and -MIWI2 expression vectors were kindly gifted by Dr. Kuramochi-Miyagawa (Kojima et al. 2009). Immunofluorescence Adult marmoset testes were fixed in 4 % (w/v) paraformaldehyde overnight at 4 °C. Testes were the soaked in 15 % and 30 % (w/v) sucrose sequentially. Testes embedded in OCT compound (Sakura Finetech, 4583) or SCEM (Section-Lab, SCEM) were cut into 7 µm sections using cryostat (Leica, CM3050S). After air-drying, sections were placed in Target Retrieval Solution (Dako, S1699) and subjected to antigen retrieval using autoclave at 105 °C for at least 5 min and then blocked for 1 hour at room temperature in 2 % Block Ace (DS Pharma Biomedical, UK-B40). Primary antibody incubation was done overnight at 4 °C in blocking reagent with 0.1 % (v/v) Triton X-100. After washing in PBS, the appropriate secondary antibody (1:1000 dilution, Alexa Fluor series, Molecular Probes) and 4’,6-diamidino-2-phenylindole (DAPI) solution (1:10000 dilution; Dojindo, 340-07971) was added and incubated at room temperature for 1 hour in blocking reagent with 0.1 % Triton X-100. Sections were mounted on glass slides in PermaFluor Aqueous Mounting Medium (Thermo, TA-006-FM). Confocal laser microscopy (Zeiss, LSM710) was used to acquire images. The following primary antibodies were used: cultured supernatant of mouse IgG1 and IgG2b monoclonal anti-MARWI hybridoma cells (1:50 dilution, 2D9 and 4A1 line, respectively), rabbit polyclonal anti-DDX4 (VASA) (1:100 dilution for marmoset and 1:1000 dilution for mouse, Abcam, ab13840), rabbit polyclonal anti-DAZL (1:200 dilution for marmoset and 1:1000 dilution for mouse, abcam, ab34139), mouse IgG1 monoclonal anti-phospho-Histone H2A.X (Ser139) (γH2AX) (1:500 dilution, Millipore, 05-636). Western blotting Western blot analysis was performed as described previously (Miyoshi et al. 2010). Ten µg of protein from tissue samples or immunoprecipitates was subjected to SDS-PAGE. Culture supernatants of anti-MARWI (1A5) hybridoma cells (1:10 dilution), mouse monoclonal anti-FLAG M2 (1:2000 dilution, Sigma, F1804) and mouse monoclonal anti-β-TUBULIN (1:5,000 dilution, DSHB, E7) were used. Immunoprecipitation Immunoprecipitation details have been described previously (Saito et al. 2009). Briefly, fragmented testes tissues were homogenized in binding buffer (30 mM HEPES, pH 7.3, 150 mM potassium acetate, 2 mM magnesium acetate, 5 mM dithiothreitol (DTT), 0.1% Nonidet P-40 (Calbiochem, 492016), 2 mg/mL Leupeptin (Sigma, L9783), 2 mg/mL Pepstatin (Sigma, P5718) and 0.5% Aprotinin (Sigma, A6279)). MARWI and MIWI proteins were immunopurified using anti-MARWI (1A5) immobilized on Dynabeads protein G (Life Technologies, 10004D). Reaction mixtures were incubated at 4°C for 3 h and beads were rinsed twice with binding buffer then five times with wash buffer (binding buffer with 0.5 M NaCl). β-elimination Periodate oxidation and β-elimination was performed as described (Kirino and Mourelatos 2007; Ohara et al. 2007; Saito et al. 2007; Simon et al. 2011). A 100 µL mixture consisting of purified RNAs and 10 mM NaIO4 (Wako, 199-02401) was incubated at 4°C for 40 min in the dark. The oxidized RNAs were then incubated with 1 M Lys-HCl at 45°C for 90 min to achieve β-elimination. Northern blotting Northern blot analysis was carried out essentially as previously described (Saito et al. 2010). Total RNAs of testis or MARWI immunoprecipitates were isolated using ISOGEN (NIPPON GENE, 311-02502), run on 12% acrylamide-denatured gels, and transferred onto Hybond N+ or N membranes (GE Healthcare, RPN303N or RPN303B, respectively). RNAs were crosslinked with 280 nm UV or 1-ethyl-3-(3-dimenthylaminopropyl) carbodiimide (EDC)-mediated chemical crosslinking methods (Pall and Hamilton 2008). The hybridizations were performed at 42°C in 0.2 M sodium phosphate (pH 7.2), 1mM EDTA (pH8.0) (Life Technologies, 15575-038) and 7% SDS, and washed at 42°C in 2 × saline sodium citrate and 0.1% SDS. The sequence of northern probes is described in Supplemental Table S10. Preparation of small RNA libraries Preparation of MARWI-piRNA and total small RNA libraries used the NEBNext Small RNA Library Sample Prep Set (New England BioLabs (NEB), E7330) with slight modifications. Gel-purified small RNAs obtained using the Small RNA Gel Extraction kit (TaKaRa, RR065) underwent 3’ ligation at 16°C for 18 hour, then free 3’ adaptors were degraded using 5’ deadenylase (NEB, M0331) and RecJf (NEB, M0264). 3’ ligated RNAs underwent 5’ adaptor ligation at 16°C for 18 hour. RNAs were reverse transcribed using SuperScriptIII (Life Technologies, 18080-044) and were PCR-amplified using LongAmp Taq 2xMaster mix (NEB, M0287) with 18 cycles. In vitro Slicer assay The in vitro slicer assay was performed as described previously (Miyoshi et al. 2008). 32P-radio labeled piR-2 target RNAs were incubated with MARWI immunoprecipitates at 35°C in cleavage buffer (25 mM Hepes-KOH (pH 7.5), 50 mM KOAc, 5 mM Mg(OAc)2, 5 mM DTT, and 0.4 unit RNasin plus (Promega, N2611)) for 4 hour. The sequence of piR-2 target RNA is described in Supplemental Table S10. Comparison of two replicate samples for RNA-seq and MARWI-IP small RNA -seq To analyze MARWI associated piRNAs and total RNA, we generated two libraries from two different samples (51 month- and 41 month-old adult marmoset testis). Expression level of each gene was calculated for each of the libraries and plotted as dot plot for RNA-seq (R2 = 0.50), where the number of reads obtained from each unique sequence was counted for each of the libraries and plotted as a dot plot for MARWI-IP small RNA-seq (R2 = 0.89). If the sequence is only present in one of the libraries, the read count was considered to be zero for the other library. Annotation of the reads The annotation of marmoset genome mapped reads was determined as previously described (Kawamura et al. 2008). Briefly, the overlap between mapped regions of the reads was examined using feature track data of the UCSC Genome Browser (Meyer et al. 2013) and Ensembl databases (Flicek et al. 2013). Reads were assigned to a feature when the length of its overlap was longer than 90% of the small RNA. We defined the priority of the feature assignment to avoid any conflict of assignment. The priority was in the following order: miRNA, ncRNA, rRNA, tRNA, snRNA, snoRNA, to remove small RNA contaminants, protein coding genes, transposons, and repeats. The assignments to miRNA, ncRNA, rRNA, snRNA, snoRNA, and coding genes were performed using Ensembl tracks (Flicek et al. 2013). We used UCSC Genome Browser tRNA tracks for tRNAs, and UCSC Genome Browser RepeatMasker (repeats) and Simple repeats tracks for repeats (Meyer et al. 2013; Smit et al. 2010). Transposons were defined by a combination of Ensembl retrotransposed tracks (Flicek et al. 2013), and the RepeatMasker track (transposon) from the UCSC Genome Browser (Meyer et al. 2013; Smit et al. 2010). RNA-seq and data processing RNA-seq libraries were prepared with the directional mRNA-seq library prep protocol using the TruSeq Small RNA Sample Prep Kit (Illumina, RS-200-0012) by TaKaRa Dragon Genomics. Two replicate libraries were prepared and sequenced using HiSeq2000, and obtained reads were combined resulting in total of 168,398,076 reads. Reads were mapped to the marmoset reference genome (calJc3, WUSTL 3.2) using Bowtie (Langmead et al. 2009) with default parameters. Expression of each transcript was determined using TopHat (Trapnell et al. 2009) and Cufflinks (Trapnell et al. 2010), except for Argonaute proteins as these sequences were unavailable within the Ensembl data (C_jacchus3.2.1)(Flicek et al. 2013) (see below). Calculation of RPKM for Argonaute/Piwi expression Because of the lack of marmoset Argonaute/Piwi mRNA sequence in Ensembl and GenBank databases (Benson et al. 2013; Flicek et al. 2013), we determined the expression of marmoset Argonaute/Piwi mRNAs by calculating the number of reads that uniquely map to the transcript sequence of each Argonaute/Piwi mRNA. These mapped reads were normalized by the length of each transcript and the total number of genome mapped reads to calculate Reads per Kilobase per Million mapped reads (RPKM). Sequence logo and heatmap We used motifStack bioconductor package to produce sequence logos. Sequences were aligned to the 5’ end to search for nucleotide bias. Heatmaps were depicted using Java TreeView software (Saldanha 2004). Distribution of RNA-seq and small RNA-seq data RNA-seq and MARWI-IP small RNA-seq distribution was determined using the reads that were mapped 1–5 times within the genome. We normalized the reads by dividing the number of mapped reads by the number of positions mapped to the genome. Regions with genomic sequences consisting of unidentified nucleotides (N), are shown as gaps. Transposons, coding genes, and pseudogenes were defined using annotations from the Ensembl database (Flicek et al. 2013), UCSC Genome Browser (Meyer et al. 2013), and manual curation of the pseudogenes according to the host gene sequence. Some piRNA clusters were merged within the MARWI-IP small RNA-seq distribution since the gaps within the marmoset genomic sequence seem to have divided the single cluster into two. miRNA prediction We used total small RNA-seq reads 21–23-nt in length to predict miRNAs (1,258,550 reads).
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