Loss of l(3)mbt leads to acquisition of the ping-pong cycle in Drosophila ovarian somatic cells

Sumiyoshi et al. show that CRISPR-mediated loss of function of lethal (3) malignant brain tumor [l(3)mbt] leads to ectopic activation of the germ-specific ping-pong cycle in ovarian somatic cells. Perinuclear foci resembling nuage, the ping-pong center, appeared following l(3)mbt mutation.


Fly stock, cell culture and RNAi
Oregon-R was maintained at 25°C. Δmbt-OSCs and OSCs were grown at 26°C in culture medium prepared from Shields and Sang M3 Insect Medium (Sigma) supplemented with 0.6 mg/mL glutathione, 10% FBS, 10 mU/mL insulin, and was amplified by RT-PCR from OSC total RNA using gene-specific primers. It should be noted that the 5' end of each primer contained the T7 RNA polymerase promoter sequence. PCR products were purified using the FastGene Gel/PCR Extraction Kit (NIPPON Genetics) according to the manufacturer's instructions. Purified PCR products were used to produce dsRNAs using a T7-Scribe Standard RNA IVT Kit (CELLSCRIPT). The resultant RNAs were purified according to the manufacturer's instructions, heated for 5 min at 95°C, and then left to cool to room temperature. Then, 5 × 10 6 cells were suspended with 5 µg of dsRNA and incubated for 4 days at 26°C. The siRNAs and PCR primers used in this study are summarized in Supplemental Table S3.

Plasmid constructs and OSC transfection
Construction of the Myc-Aub, Myc-AGO3, and Myc-EGFP vectors used in this study was described previously (Sato et al. 2015). To construct a Myc-Vasa expressing plasmid, a full-length vasa cDNA was amplified by RT-PCR and subcloned into pAcM under the control of the actin 5C promoter.

Immunoprecipitation
Immunoprecipitation was performed as previously described (Sato et al. 2015).
In brief, immunoprecipitation of Piwi and Aub complexes from Δmbt-OSCs was performed using anti-Piwi (Saito et al. 2006) and anti-Aub (Nishida et al. 2007) antibodies in NP40 buffer. Immunoprecipitation of AGO3 complexes was performed using an anti-AGO3 antibody (a kind gift from Dr. Dahua Chen, Chinese Academy of Sciences, China) in RIPA binding buffer.

qRT-PCR
qRT-PCR was performed as previously described (Sato et al. 2015). In brief, total RNAs were isolated using ISOGEN (Nippon Gene) according to the manufacturer's instructions. Total RNAs were treated with DNase to eliminate DNA contamination. Total RNA (1 µg) was annealed with an oligo-dT primer and reverse transcribed using a Transcriptor First strand cDNA Synthesis Kit (Roche) according to the manufacturer's instructions. The resulting cDNAs were amplified with StepOnePlus (Applied Biosystems) using SYBR Premix Ex Taq (TaKaRa). The primer sets used are shown in Supplemental Table S1. The amplification efficiency of a qPCR reaction was calculated based on the slope of the standard curve. After confirming the amplification efficiency values (between 95% and 105%), relative steady-state RNA levels were determined from the threshold cycle for amplification.

Visualization of small RNAs
Visualization of small RNAs was performed as previously described (Sato et al. 2015). In brief, for RNA labeling with radioisotopes, total RNAs and immunopurified RNAs were dephosphorylated with CIP (NEB), and then labeled with γ-32 P-ATP using T4 polynucleotide kinase (TaKaRa). The radiolabeled RNAs were separated on a denaturing polyacrylamide gel. The signal was detected using a Typhoon FLA 9500 (GE Healthcare).

Preparation of total RNA libraries and bioinformatic analysis
rRNAs were removed from isolated total RNAs using a Ribo-Zero rRNA Removal Kit (Human/Mouse/Rat) (Illumina). Total mRNA libraries were prepared using a TruSeq Stranded mRNA HT Sample Prep Kit (Illumina) according to the manufacturer's instructions and sequenced using a HiSeq 2500 (Illumina).
Paired-end sequence reads were split into forward and reverse reads and mapped separately on the Drosophila genome (Dm3, BDGP Release 5), and then annotated to Drosophila genes defined in the UCSC Genome Browser (FlyBase) using Cuffcompare. Differential expression analysis of mRNA-seq expression profile was performed using edgeR package in R. The lists of differentially expressed genes (p < 0.05) were applied for functional annotation (gene ontology) analysis using DAVID. Paired-end sequence reads were also mapped to the transposon consensus sequences (Senti et al. 2015). To compare mRNA abundance between two libraries, FPKM (fragments per kilobases of exons per million mapped reads) normalization was performed.

Preparation of small RNA libraries and bioinformatic analysis
Cloning and analysis of small RNA libraries were performed essentially as previously described (Sato et al. 2015 Sense/antisense strand bias, sequence logo, and ping-pong signature on each transposon or all transposons were analyzed as previously described (Sato et al. 2015). To calculate ping-pong Z 10 scores, overlaps at position 1-9 and 11-25 nt were used as background. Phasing analysis was performed as previously described (Han et al. 2015). The score for a 3'-to-5' distance was calculated by Σminimal (Mi, Ni+x); Mi is the number of upstream reads whose 3' ends are located at position i, and Ni+x is the number of downstream reads whose 5' ends are located at position i+x. To calculate the ping-pong Z 1 score, overlaps at position 0 and 2-50 nt were used as background.

Deep sequencing datasets have been deposited in the DDBJ Sequence Read
Archive under the accession number DRA004265.   Table S1. List of Gene Ontology terms of the biological process category associated with differentially expressed genes in OSCs and Δmbt-OSCs.

Supplemental Table S2. List of differentially expressed genes in OSCs and
Δmbt-OSCs.