alias: E-MTAB-7390:sHSCc_p
title: Illumina HiSeq 2000 paired end sequencing; 3'-Seq sHSC/pHSC
design: 3'-Seq sHSC/pHSC
library_construction_protocol: 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. HSC cells  were identified by immunophenotype (Lineage- Kit+ CD150+ CD48- CD34-), purified by FACS and subsequently subjected to 3'-seq.  Mice were injected intraperitoneally with 50 µg pIC or PBS alone. 16h later, mice were sacrificed and bone marrow cells were used for subsequent experiments. 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))
identifiers.namespace: University of Heidelberg
identifiers.id: E-MTAB-7390:sHSCc_p