title: Illumina HiSeq 2000 paired end sequencing; 3'-Seq of multipotent progenitor cells MPP5
design: 3'-Seq of multipotent progenitor cells MPP5
library_construction_protocol: Bone marrow (BM) was isolated from pooled femora, tibiae, ilia and vertebrae of C57BL6 mice by gentle crushing in PBS using a mortar and pistil. MPP5 progenitors were identified by immunophenotype (Lineage- Sca1+ Kit+ CD150- CD48- CD34+ CD135-), purified by FACS and subsequently subjected to 3'-seq. 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))