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Mx1Cre, (M, 2) Mx1Cre;p53loxp/ loxp , (MP, 2) Mx1Cre;LSLKrasG12D, (MK, 3)

RNA seq to understand mechanisms underlying transformation/self-renew of myeloid progenitor cells with KrasG12D activation and p53 deletion. Mx1Cre, (M, 2) Mx1Cre;p53loxp/ loxp , (MP, 2) Mx1Cre;LSLKrasG12D, (MK, 3) Mx1Cre;p53loxp/loxp;LSLKrasG12D, (MPK, 3 ). pIpC.

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Mx1Cre, (M, 2) Mx1Cre;p53loxp/ loxp , (MP, 2) Mx1Cre;LSLKrasG12D, (MK, 3)

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  1. RNA seqto understand mechanisms underlying transformation/self-renew of myeloid progenitor cells with KrasG12D activation and p53 deletion Mx1Cre, (M, 2) Mx1Cre;p53loxp/loxp, (MP, 2) Mx1Cre;LSLKrasG12D, (MK, 3) Mx1Cre;p53loxp/loxp;LSLKrasG12D, (MPK, 3) pIpC FACS sorting for LinlowCkit+Sca1- myeloid progenitor cells (doubly sorted 5,000-20,000 cells) RNA extraction (1.5-2 ng) Ovation® RNA-SeqSystem (to prepare amplified cDNA fromas little as 500 pg total RNA ) Library generation Sequencing (36bp single end)

  2. Multiple analyses of RNA seq data Cleaned up reads Reference sequence De novo assembled transcriptome Ref Flat Ref Seq Reference sequence HTSeq Cufflink Annotation E D C CC Differential expression analysis Annotation EdgeR DeSeq Cuffdiff Comparison was done for: MPK/MK, MPK/M, MK/M DEA On going

  3. Overlapping of multiple analyses results MK/M MPK/MK 414 395 MPK/M 437 116 18 41 205 29 3 90 23 234 202 16 32 107 94 245 EdgeR 35 DeSeq 18 118 Cuffdiff Log2(fold change) ≥1 and P≤ 0.2

  4. Top 5 functional networks interfered in MPK vs MK comparison

  5. Candidate genes to validate • Proof of principle by individual qPCR? • 17 genes • Large scale by qPCR chip? • Effects of p53 loss on top of Kras mutation • p53 targets • Interesting functions • Inflammation/immune response • Cellular interaction/signaling • Hematopoietic development [MPK/MK (41 genes)  MPK/M (245 genes)](9 genes)  MPK/MK (41 genes)  MK/M (234 genes)](10 genes) [MPK/M (245) - MK/M(234)](117 genes) MPK/MK (41 genes)= 151 genes [MPK/MK (30) MPK/M(38)](58 genes) If  above 151 genes =208 genes

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