title: RNA-seq of mouse hematopoietic stem cell and multipotent progenitor cell populations
design: RNA-seq of mouse hematopoietic stem cell and multipotent progenitor cell populations
library_name: mpp6-epcrneg-5_p
library_construction_protocol: To prepare for BM cell suspension, bones were crushed and incubated with ACK buffer for 5 minutes at room temperature to remove excess red blood cells. Lineage depletion was performed using the EasySep Mouse Hematopoietic Progenitor Cell Isolation Kit (STEMCELL technologies, #19856) according to the manufacturer's instruction. The Lin-depleted cells were then stained with the following antibodies: BV711 anti-mouse c-Kit (clone 2B8; BioLegend; 105835) or BUV395 anti-mouse c-Kit (clone 2B8; BD Biosciences; 564011), APC-Cy7 anti-mouse Sca1 (clone D7; BD Biosciences; 560654), PE anti-mouse EPCR (clone eBio1560; eBioscience; 12-2012-82), FITC anti-mouse CD34 (clone RAM34; eBioscience; 11-0341-82) or eFluor 660 anti-mouse CD34 (clone RAM34; eBioscience; 50-0341-82), PE-Cy7 anti-mouse CD48 (clone HM48-1; BioLegend; 103424), BV421 anti-mouse CD135 (clone A2F10.1; BD Biosciences; 562898) or APC anti-mouse CD135 (clone A2F10; BioLegend; 135310),PE-Cy5 anti-mouse CD150 (clone TC15-12F12.2; BioLegend; 115912) or BV785 anti-mouse CD150 (clone TC15-12F12.2; BioLegend; 115937), BUV496 anti-mouse CD16/32 (clone Ab93; BD Biosciences; 751694), Alexa Fluor 700 anti-mouse CD4 (clone GK1.5; eBioscience; 56-0041-82),Alexa Fluor 700 anti-mouse CD8a (clone 53-6.7; eBioscience; 56-0081-82), Alexa Fluor 700 anti-mouse CD11b (clone M1/70; eBioscience; 56-0112-82),Alexa Fluor 700 anti-mouse GR1 (clone RB6-8C5; eBioscience; 56-5931-82), Alexa Fluor 700 anti-mouse B220 (clone RA3-6B2; eBioscience; 56-0452-82), Alexa Fluor 700 anti-mouse Ter119 (clone TER-119; BioLegend; 116220).  All antibody staining were performed at 4 °C for approximate 30 minutes. Cells were then washed to remove unbound antibodies and resuspended in FACS buffer containing 7-AAD (BD Biosciences; 51-68981E) prior to cell sorting using BD FACSAria-II/III, BD Fusion or BD Symphony S6 (BD Biosciences). Between 400 – 2000 cells from each HSPC populations were sorted. Cells were sorted into extraction buffer and total RNA isolation was performed using the ARCTURUS PicoPure RNA isolation kit (Life Technologies, Invitrogen) following the manufacturer's instruction, with DNase treatment using the RNase-Free DNase Set (Qiagen).  RNA qualification and quantification was performed using the bioanalyzer and samples with RIN >=7.5 were used to for cDNA library preparation using the New England Biolabs (NEB) Low Input RNA kit following the manufacturer's instruction. 320 pg of total RNA for each sample was used as starting material.  Amplification of cDNA was performed using 17 PCR cycles. 