title: GSM5050975: MPP_GCSF; Mus musculus; RNA-Seq
library_construction_protocol: Whole Bone marrow was isolated from femur, tibia, hip and vertebrae via gentle crushing using a mortar and pestle. Stem and progenitor cells were enriched via lineage depletions (Miltenyi Biotech, 130-090-858). For treatment with niche stimulants cells were FAC-sorted  into the following populations: HSCs (Lin-, c-Kit+, Sca1+, CD48-, CD150+, CD34-), MPP1s (Lin-, c-Kit+, Sca1+, CD48-, CD150+, CD34+), MPPs (Lin-, c-Kit+, Sca1+, CD48-, CD150-), MPP2s (Lin-, c-Kit+, Sca1+, CD48+, CD150+), and MPP3/4s (Lin-, c-Kit+, Sca1+, CD48+, CD150-). Sorted MPPs were each labeled with different hashtag oligonucleotide (HTO) conjugated antibodies (Biolegend, TotalSeq™-A Cell Hashing). After 30min incubation cells were washed and the four MPP samples were pooled at equal ratios into a single MPP sample per condition (control, dmPGE2, pIC, GCSF, indo) for library preparation. scRNA-Seq: Library for HSC_Repl1 were processed according to manufacturer's recommendations (10X Genomics V2, CG00052 Rev B). Libraries were sequenced on a Next-seq 500 using a 75 cycle kit. Libraries for stimulated HSC and MPP scRNA-Seq samples were processed according to manufacturer's recommendations (10X Genomics V3, CG000185 Rev B) through Post Gem-RT Cleanup – Dynabeads (step 2.1). At the cDNA amplification step (Step 2.2) an HTO additive primer was added to the amplification mix and steps 2.3A and 2.3B (using 70 µL of SPRI beads) were followed exactly to separate HTOs from cDNAs. cDNA library preparation was finished according to 10x Genomics recommendations. Purified HTO fraction was amplified using SI PCR primer and TruSeq D70x_s indexes according to HTO antibody manufacturer instructions (Biolegend). scATAC-Seq: For ATAC-seq nuclei were isolated from HSCs and MPPs (pooled MPP, MPP1, MPP2 and MPP3/4) according to manufacturer's recommendations (CG000169 Rev D - Low Cell Input Nuclei Isolation). ScATAC-Seq libraries were prepared using manufacturers recommendations (10x Chromium Single Cell ATAC , CG000168 Rev B). ScRNA-Seq libraries form niche stimulation experiment and scATAC-Seq libraries were sequenced on a NOVAseq 6000 using a 100 cycle kit.