title: Illumina HiSeq 2000 paired end sequencing; RNA-Seq of osteoclasts derived from female Phlpp1 cKO or control littermates
design: RNA-Seq of osteoclasts derived from female Phlpp1 cKO or control littermates
library_construction_protocol: Samples were placed on ice, rinsed with PBS and lysed in Trizol. Hind limbs were dissected and bone marrow macrophages were collected from female 6 to 8-week-old Control, Phlpp1 cKOCtsk or Phlpp1-/-  mice as previously described (16). Briefly, cells were flushed, pelleted and red blood cells were lysed (RBC Lysis Buffer, #00-4333-57, Invitrogen, Carlsbad, CA). Cells were pelleted and cultured overnight in phenol red-free alpha MEM, 10% FBS and 1% antibiotic/antimycotic supplemented with 35 ng/mL M-CSF (#416-ML, R&D, Minneapolis, MN).  Non-adherent cells were then placed in culture medium supplemented with 35 ng/mL M-CSF, and 90 ng/mL RANKL (#315-11, Prepro Tech, Rocky Hill, NJ).  Cultures were fed on day 3 with culture medium plus 35 ng/ml M-CSF, and 90 ng/mL RANKL Trizol extraction Random hexamers were used to generate cDNA libraries via reverse transcription
attributes.tag: Experimental Factor: genotype
attributes.value: Phlpp1 cKO