Cell growth, fractionation, RNA isolation

1. Cell growth, fractionation, RNA isolation

2. Cell growth conditions • Growth to 600,000-800,000 cells/ml • Media RPMI1640 + 10% FBS + pen-strep • Subcellular fractionation done on fresh cells • Typically 50-70% of cells in S-phase as assessed by the FACS analysis

3. Cyto-nuclear fractionation • Cells are lysed for 5 minutes on ice in the RLN buffer containing 50 mM Tris pH 8.0; 0.5% Igepal (NP40 substitute); 140mM NaCl and 1.5 mM MgCl2. Nuclei are spun down at ~500g for 5 minutes. • Supernatant becomes the cytosolic fraction • The nuclear pellet is washed once with the RLN buffer & spun. • Wash is discarded. The pellet becomes the nuclear fraction.

4. RNA isolation: K562 cyto and nuclear • Short (Cyto and nuclear) • 1. Purification on Qiagen RNA/DNA columns • Get rid of DNA and RNAs > 200nt • 2. DNAse treatment + phenol/chlorophorm • 3. For some samples, PAGE-based separation into three fractions at CSHL • Additional step to get rid of RNAs > 200nt • Long Nuclear • 1. Spin of extract through the CsCl cushion • Get rid of DNA and RNAs < 200nt • 2. Clean-up on Rneazy • Get rid of RNAs < 200nt • 3. Fractionation into A+ & A- • 4. DNAse treatment of A+ & A- followed by cleanup on Rneazy • Long Cyto • 1. Purification on Rneazy • Get rid of RNAs < 200nt • 2. Fractionation into A+ & A- • 4. DNAse treatment of A+ & A- followed by cleanup on Rneazy • Each procedure incorporates at least two different methods to get rid of DNA (columds + DNAse for short RNAs; CsCl + DNAse for long RNAs) and two different methods of separation (columns + gel for short RNAs and CsCl + columns for long RNAs).

5. Profiles of K562 cyto and nuclear RNAs used for experiments B1 K562 cyto A+: diTags B4 K562 cyto A+: arrays B1 K562 cyto A-: CAGE, Arrays B3 K562 cyto short: used for library construction B3 K562 nuclear short: used for library construction B4 K562 nuclear A-: CAGE, Arrays B4 K562 nuclear A+: diTags, Arrays (1:1 with B5) B5 K562 nuclear A+: Arrays (1:1 with B4)

6. Isolation of subcompartments Polysomes, chromatin and nucleolus

7. RNP 40S 60S 80S Polysomal fraction Polysomal profile of K562 on the 20-60% sucrose gradient. Absorption at 254 nm bottom top

8. Polysomal RNA preparation Separation of polysomal fraction on sucrose gradient Preparation of total polysomal RNA using trizol Fractionation of total into long and short using miRvana Fractionation of long into polyA+ and polyA- Further purification of short using Qiagen RNA/DNA columns (miRvana did not work 100% for this fraction). K562 polysomal A- RNA: CAGE, arrays K562 polysomal short RNA: sequencing

9. Scheme of the cell fractionation process to purify chromatin Resuspended in RSB buffer and disrupted nuclei by sonication Cells Cell Disruption Layered over 30% sucrose in NaCl-Tris buffer and centrifuged at 5000g/15min Centrifugation 2300rpm/6min Nuclear Supernatant 1 Nucleoli Layered over 60% sucrose in NaCl-EDTA-Tris Buffer and centrifuged at 27000g/1.5hr Cytoplasm Nucleus Nuclear Supernatant 2 Chromatin (Purified chromatin) Nuclei purification Chromatin purification Jaswant Bhorjee and Thoru Pederson Biochemistry. 1973 Jul 3;12(14):2766-73.

10. Summary of the isolated subcellular compartments using our protocol Cells Cytoplasm Nucleus Nuclear supernatant 1 Nucleolus Nuclear supernatant 2 Chromatin

11. Western blot for protein markers of subcellular compartments K562 Nucleus Total cell Cytoplasm Nucleolus Nu super 2 Chromatin Nu-super 1 Cytoskeleton marker β-Tublin Nucleolus marker Fibrillarin Chromatin Marker Histone 3 Not a loading issue hnRNP C1 + C2 hnRNP C1 + C2 is a major component of nuclear matrix

12. Conclusions • Chromatin is free from nucleolus based on fibrillarin results, but nucleolus has chromatin contamination based on the H3 western • Supernatant 2 is free from nucleolus and chromatin. Its composition is not clear, may be a sum of nuclear matrix, nucleoplams and perhaps other nuclear bodies • More controls are needed to trace the fate of other nuclear parts

13. SnoRNAs are enriched in purified nucleolus associated RNA snoRNA U70 Nucleolus-RNA short Chromatin-RNA short Nucleolus-RNA long Chromatin-RNA long Short RNA: direct labeling, 2 ug RNA, Encode v2 F Long RNA: cDNA,10ug RNA, Encode v2 R

14. SnoRNAs are enriched in purified nucleolus associated RNA snoRNA ACA36 snoRNA ACA56 Nucleolus-RNA short Chromatin-RNA short Nucleolus-RNA long Chromatin-RNA long

15. Agilent bioanalyzer profile of K562 purified RNA (mirVana) Long Nucleolar RNA Long Chromatin RNA Short Nucleolar RNA Short Chromatin RNA

16. Labeling and microarray hybridization • Final RNA (polyA-, polyA+ or short) is always treated with DNAse before sending or labeling • RNA is converted into ds-cDNA using random hexamers • Three independent labelings (starting from cDNA synthesis) are done by three individuals on the same pool of RNA to assess the reproducibility • The three independent replicas are hybridized to ENCODE arrays in duplicates. Typicall correlations are on the order of > 0.9 with the lowest of 0.88 on the level of graph data • The labelings are then pooled and hybridized to the 91-array set • 10 ug and 2.5 ug of ds-cDNA from polyA- and polyA+ RNAs respectfully are hybridized per microarray

17. Control transcripts XIST in the nuclear fraction

18. XIST locus in K562: graphs scaled by intensity (1-15,000) Cyto A+ Cyto A- Nuclear A+ Nuclear A- XIST

19. XIST locus in K562: graphs scaled by percentile (50th-99.9th) Cyto A+ Cyto A- Nuclear A+ Nuclear A- XIST XIST

20. Control transcripts mRNAs in the polysomal fraction

21. A typical protein coding gene (IRAK1) enriched in polysomal A- fraction Polysomal A- Cytosolic A-

22. Un-expected results Long, presumably non-coding RNAs in the polysomal fraction?

23. Large intergenic region on chr. 11 enriched in polysomal polyA- fraction Polysomal A- Cytosolic A-

24. Completed whole-genome maps • K562 • Long cytosolic polyA+ • Long cytosolic polyA- • Long nuclear polyA+ • Long nuclear polyA- • Long polysomal polyA- • Prostate • Long polyA+ • Long polyA-

25. Issues • Which regions are enriched in a compartment ? • May be relatively straightforward. For example, as simple as making a ratio difference map between different compartments. • A more difficult question: which regions should be chosen for further characterization? • Presence of large regions of nuclear transcription in introns and also often in the regions of extended alternative TSSs. How to separate heteronuclear RNA from functional stand alone transcripts? Perhaps the heteronuclear RNA is also functional: may be the function of some of the extended TSSs is to transcribe large regions of the genome?

26. Hemoglobin locus. chr11: 5,198,743 - 5,501,015 Cyto A+ Nucl A+ Ratio of Nucl A+ vs Cyto A+ nuclear-enriched transcript(s) Extended TSS

27. Two TSSs nuclear-enriched transcript(s) TEAD1 locus. chr11: 12,636,596 - 12,938,868 Cyto A+ Nucl A+ Ratio of Nucl A+ vs Cyto A+

28. A nuclear enriched transcript in gene desert region: chr21: 15244343-15377412 Cyto A+ Nucl A+ Ratio of Nucl A+ vs Cyto A+

29. Extended TSS Or is it a gene desert? Cyto A+ Nucl A+ Ratio of Nucl A+ vs Cyto A+

30. Nuclear-enriched transcripts with different abundances MLSTD2 locus: chr11: 13,641,847 - 13,715,855 Cyto A+ Nucl A+ Ratio of Nucl A+ vs Cyto A+

31. Nuclear-enriched transcripts with different abundances MRPS6 locus: chr21: 34,359,111 - 34,450,983 Cyto A+ Nucl A+ Ratio of Nucl A+ vs Cyto A+