1. Stefan Aigner Christian Carson Rusty Gage Gene Yeo Crick-Jacobs Center Salk Institute Analysis of Small RNAs in Stem Cell Differentiation

2. Neuronal specification Neural progenitor (NP) Embryonic stem (ES) Neuron (Ne) Oct4 DAPI ES Nestin Sox1 DAPI NP Map2a+b Tuj1 DAPI NE

3. Cloning of 18-30 nt small RNAs 18 nt 24 nt 30 nt SybrGold stained 15% acrylamide/urea gel ES NP Adapter ligation RNA Reverse transcription, PCR dsDNA Cloning and Sequencing Mapped to repeat-masked human genome (>18bp) ES 1.04M NP 2.46M Ne 1.83M

4. ~4M reads clustered into ~76,000 non-redundant clusters 81% of known mature miRNAs detected ~16K reads per pre- miRNA on average 30%

5. mir-302d 6323 reads Sequence-level resolution

6. Classifier for novel hairpins (MIRESQUE) 315 of 325 (96%) known microRNA hairpins can be detected reliably 215 MIRESQUE predicted miRNA hairpins (out of 2688 unlabeled samples) chrX:113772628_3pchr15:81221813_5pchr22:18448149_5pchr19:58867025_5p 35 30 24 18 ESNP ESNPESNPESNP nt 5S rRNA ESNP let-7c

7. Summary of miR predictions 80% of known 430 848674 113

8. High resolution allows us to correct annotations 5p3p ‘passenger’‘guide’ 24 “Guide” vs “passenger” strand changes ‘target’ predictions.

9. Correcting 5’ ends of microRNAs 49 known miRNAs have incorrectly annotated 5’ ends Mir-34b GTGGTGGTTAcaatcactaactccactgccatCAAAACAAGG Edges..........>>>------------------<<<<....... +ES AATCACTAACTCCACTGCCAT 1 +ES AATCACTAACTCCACTGCCATC 2 +ES ATCACTAACTCCACTGCCATCA 2 +NP CAATCACTAACTCCACTGCCATCAA 1 +NP CAATCACTAACTCCACTGCCAT 1 +NP ATCACTAACTCCACTGCCATCA 1.5 +NE AATCACTAACTCCACTGCCATC 62 Mir-34b*TAAGAAAAGAtcgtgcatccctttagagtgttACTGTTTGAG Edges........>>>->--------------<<<<<.......... +ES GATCGTGCATCCCTTTAGAGTGT 0.5 +ES ATCGTGCATCCCTTTAGAGTG 18 +ES ATCGTGCATCCCTTTAGAGTGTT 2.5 +ES ATCGTGCATCCCTTTAGAGTGT 264 +ES TCGTGCATCCCTTTAGAGTGT 14 +ES TCGTGCATCCCTTTAGAGTGTT 0.5 +ES TCGTGCATCCCTTTAGAGTG 2 +ES TCGTGCATCCCTTTAGAGT 2.5

10. Sense/antisense microRNAs Known example: mir-338 11 annotated mirs have s/as pairs ES NP NE

11. Switching strand preferences ES NP NE 13 microRNAs with strand switches Khvorova et al, Cell 2003 Schwarz et al, Cell 2003

12. C/D and H/ACA snoRNAs processed as miRNAs? ES NP NE

13. Digital expression reveals hES/hNP-specific miRs ESNENP ESNENP ESNENP ESNENP ES NE NP

14. Small RNA summary ES, NP, NE express an unexpectedly complex set of small non-coding RNAs whose biological significance remains to be investigated. MIRESQUE designed: We discovered hundreds of novel bona fide microRNAs, dozens of which are in coding regions of genes. High resolution allows correct guide/passenger annotations, correct 5’ ends of miRs, Sense/antisense microRNAs were identified MicroRNAs that switch strands The microRNA profiles of ES, NP, NE are distinct. Novel ES-specific miRs.