(A) Estimate of false-positive/negative rate of the in situ screen using comparison with the independent transcriptomics data. A gene was classified as falsely positive if it was annotated as ubiquitous or specific by FISH but was not detectable by either 3Pseq or RNAseq at any time-point of oogenesis. In 20% of the experiments we failed to detect in situ signal (‘no signal’) although the transcript was detected at least at one time point by at least one deep sequencing method. These may represent false negative results, possibly due to non-functional RNA probes, however, we nevertheless included them in the downstream analysis in the no signal category. (B) mRNA enrichments in the somatic epithelial cells overlaying the oocyte (CG14639) and at the cortex of nurse cells (Actn). RNA signal shown in green. DNA, labelled with DAPI, is shown in magenta. Scale bar 30 μm. (C) CG9609 and Doa mRNAs detected in the oocyte nucleus showing the enrichment over time at stages 9, 10A and 10B. At stage 9 only few small mRNA foci are visible, at stage 10 the mRNAs were enriched in proximity of the DNA in two large foci (see arrows). (D) Karyogram showing the chromosomal position of genes for nuclear, anterior, and posterior RNA localization classes. Neither nuclear RNA genes, which often appear in foci-like enrichments nor anterior or posterior class genes appear clustered on the chromosome. (E) Examples of FISH experiment detecting distributions of non-coding RNA (in green). While pri-miRNA-318 is enriched in somatic epithelial cell nuclei, pri-miRNA-303, pri-miRNA-31-b and the long non-coding RNA CR42862 are restricted to nuclei of the germline nurse cells. Scale bar 30 μm, DNA in magenta.