Illumina NovaSeq 6000 paired end sequencing; RNA seq of various subsets of the hematopoietic system in wild type vs neutropenic mice
RNA seq of various subsets of the hematopoietic system in wild type vs neutropenic mice
Sbds -/- mice were generated using cre-mediated recombination as described previously (Wolfler, A. et al. Lineage-instructive function of C/EBPalpha in multipotent hematopoietic cells and early thymic progenitors. Blood 116, 4116-4125 (2010). Bones of the lower limbs (Hip, Femur, Ilium) were used for bone marrow (BM) and bone fraction (BF) isolation. Bones were crushed in 3x2x1mL of FACS buffer (PBS+0.5%FCS) with sterilized mortar and pestle. BM cells were pelleted by centrifuging. BM cell suspension was passed through a 40µM filter. RBCs in BM were lysed in 2mL ACK lysing buffer (Lonza) for 4 min on ice, and washed in 20mL FACS buffer. BF cells were prepared by incubating bone fragments after crushing in 2mL 0.25% Collagenase Type I (07902, STEMCELL Technologies) for 45 min in 37°C water bath, with vortexing every 15 min, washed in 20mL FACS buffer and passed through a 40µM filter again. BM and BF populations of interest were sorted, using BD FACSARIA II flow cytometer, directly into 800µl TRIzol Reagent (15596018, Thermo Fisher Scientific) and stored at -80°C until thawed for RNA extraction.  Total RNA was isolated following TRIzol manufacturer prootcol. GenElute LPA (56575, Sigma Aldrich) was used as carrier for ethanol precipitation cDNA was synthesized and amplified using SMART-Seq® v4 Ultra® Low Input RNA Kit for Illumina Sequencing (634891, Takara Bio) following the manufacturer's instructions. Amplified cDNA was further processed and the library was created according to TruSeq® Nano DNA Library Prep kit (20015964, Illumina) and paired end-sequenced (2×75 bp) on the HiSeq 2500 (Illumina)