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Stefan Aigner

Rusty Gage. Stefan Aigner. Christian Carson. Analysis of Small RNAs in Stem Cell Differentiation. Gene Yeo Crick-Jacobs Center Salk Institute. Neuronal specification. ES. NP. NE. Map2a+b Tuj1 DAPI. Oct4 DAPI. Nestin Sox1 DAPI. Embryonic stem (ES). Neural progenitor (NP).

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Stefan Aigner

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

  2. Neuronal specification ES NP NE Map2a+bTuj1 DAPI Oct4DAPI NestinSox1 DAPI Embryonic stem (ES) Neural progenitor (NP) Neuron (Ne)

  3. Cloning of 18-30 nt small RNAs RNA Adapter ligation Reverse transcription, PCR dsDNA Cloning and Sequencing ES NP 30 nt 24 nt 18 nt SybrGold stained 15% acrylamide/urea gel 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 30% 81% of known mature miRNAs detected ~16K reads per pre-miRNA on average

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

  6. Classifier for novel hairpins (MIRESQUE) ES NP ES NP ES NP ES NP ES NP nt 5S rRNA 35 30 24 18 chrX:113772628_3p chr15:81221813_5p chr22:18448149_5p chr19:58867025_5p let-7c 315 of 325 (96%) known microRNA hairpins can be detected reliably 215 MIRESQUE predicted miRNA hairpins (out of 2688 unlabeled samples)

  7. Summary of miR predictions 80% of known 848 674 430 113

  8. High resolution allows us to correct annotations 24 “Guide” vs “passenger” strand changes ‘target’ predictions. ‘guide’ ‘passenger’ 5p 3p

  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 ES Known example: mir-338 11 annotated mirs have s/as pairs 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 NE ES NP NE ES NP NE ES NP ES NP NE NP ES NP NE

  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.

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