Illumina HiSeq 2000 paired end sequencing; 3'-Seq LK Pabpn1 KD in vivo
3'-Seq LK Pabpn1 KD in vivo
Bone marrow (BM) was isolated from pooled femora, tibiae, ilia and vertebrae of C57BL6/J mice by gentle crushing in PBS using a mortar and pistil. LSK cells  were identified by immunophenotype (Lineage- Sca1+ Kit+ ), purified by FACS and  plated on ultra-low attachment plates.  Cells were cultured in 6-well ultralow attachment plates in Transduction Medium (StemPro-34 SFM, LifeTechnologies containing 50 ng/ml SCF, 25 ng/ml TPO, 30 ng/ml Flt3-Ligand (all Preprotech), 100 u/ml Penicillin/Streptomycin, 2 mM L-Glutamine, 8 µg/ml Polybrene and StemPro-34 Supplement as recommended)   shRNA-expressing plasmids (scr or Pabpn1) were purchased from Dharmacon. Viruses were packaged by cotransfection with pSPAX2 and pMD2.g into 293T cells. Viral supernatants were collected 48-hours post-transfection and viral titers determined using 293T cells. LSK cells in culture were transduced with an MOI of 35. Medium was exchanged to Complete Stem Cell Medium (specified elsewhere) 24 h later. Cells were harvested 48h after transduction and  GFP+ Cells were sorted and transplanted into irradiated recipient mice. 4 weeks after transplantation, BM was isolated and CD45.2+ GFP+ Lineage- c-Kit+ cells were sorted and used for 3'-Seq analysis. Total RNA isolation was performed using ARCTURUS PicoPure RNA isolation kit (Life Technologies, Invitrogen) according to the manufacturer's instructions. Total RNA was used for quality controls and for normalization of the starting material.   Sequencing libraries were generated with 3 ng of total RNA using the QuantSeq 3' mRNA-Seq Library Prep Kit REV (Lexogen) according to the manufacturer's indications except for adaptations (version 015UG009V0221: Do not skip step 2; 15 minute incubation time step 4; Cycle Number was adjusted to 19 using the PCR Add-on Kit for Illumina (Lexogen))
Experimental Factor: genotype
scrambled shRNA