title: Alternative platelet differentiation pathways initiated by non-hierarchically related hematopoietic stem cells
design: Alternative platelet differentiation pathways initiated by non-hierarchically related hematopoietic stem cells
library_construction_protocol: Single LIN-cKIT+ hematopoietic stem and progenitor cells generated in vivo from Vwf-tdTomato+ P-HSCs and Vwf-tdTomato- Multi-HSCs were isolated from single HSC transplanted mice 24-31 weeks after transplantation. From mice reconstituted by Vwf-tdTomato+ P-HSCs, an unbiased isolation of all CD45.1-CD45.2+ donor-derived LIN-cKIT+ cells was performed in combination with collection of index-sort information on all stained cell-surface markers (LIN, cKIT, SCA1, CD150, CD48, FLT3, CD41, CD16/32 and CD105) and reporter-gene expression (Gata1-eGFP and Vwf-tdTomato). From mice reconstituted by Vwf-tdTomato- Multi-HSCs, in addition to collection of index-sort information, a targeted selection of distinct donor-derived hematopoietic stem and progenitor cells was performed to ensure that all LIN-cKIT+ compartments were appropriately represented. No extraction was performed, instead libraries are generated directly from single-cell lysates. Single donor-derived LIN-cKIT+ cells, and internal control GMP and LSK cells, were sorted into 384-well plates with 3 uL of Smart-seq3 lysis buffer containing 0.5 U/uL RNase inhibitor (Takara), 0.1% Triton X-100 (Sigma-Aldrich), 0.5 mM dNTPs/each (Thermo Scientific), 0.5 µM Smart-seq3 oligo-dT primer (5′-biotin-ACGAGCATCAGCAGCATACGAT30VN-3′; IDT) and 5% polyethylene glycol (Sigma-Aldrich). Concentrations of dNTPs, oligo-dT primer, and polyethylene glycol were in lysis adjusted to 4 uL volume of the reverse transcription reaction. To minimize evaporation during single cell sorting, the 384-well collection plate was placed on a custom-designed aluminium 384-well adaptor (Thermo Scientific) maintained at 4ºC by the automated cell deposition unit (ACDU). Immediately after the sort, each plate was spun down and stored at -80ºC. Following a 10 min incubation at 72ºC, 1 uL of the reverse transcription mix, containing 25 mM Tris-HCl pH 8.3 (Sigma-Aldrich), 30 mM NaCl (Ambion), 1 mM GTP (Thermo Scientific), 2.5 mM MgCl2 (Ambion), 8 mM DTT (Thermo Scientific), 0.5 U/uL RNase inhibitor (Takara), 2 mM of template-switching oligo with unique-molecular barcodes (5′-biotin-AGAGACAGATTGCGCAATGNNNNNNNNrGrGrG-3′; IDT) and 2 U/uL Maxima H-minus reverse transcriptase (Thermo Scientific), was added to each well. Plates were incubated at 42ºC for 90 min, followed by 10 cycles of 50ºC for 2 min and 42ºC for 2 min, and a final incubation at 85ºC for 5 min. Generated cDNA was amplified directly after reverse transcription by adding 6 uL PCR amplification mix, containing 1X KAPA HiFi Hot Start PCR buffer (Roche), 0.5 mM MgCl2 (Roche), 0.02 U/uL KAPA HiFi Hot Start DNA polymerase (Roche), 0.3 mM dNTPs/each, 0.5 mM Smart-seq3 forward PCR primer (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATTGCGCAA*T*G-3′; IDT) and 0.1 mM Smart-seq3 reverse PCR primer (5′-ACGAGCATCAGCAGCATAC*G*A-3′; IDT) at final reaction volume of 10 uL. PCR amplification was performed by denaturation at 98ºC for 3 min, 22 cycles of 98ºC for 20 sec, 65ºC for 30 sec and 72ºC for 6 min, and final elongation at 72ºC for 5 min. After purification of amplified cDNA by home-made 22% polyethylene glycol beads at a ratio 1:0.6 sample to beads, and confirmation of appropriate library generation from randomly selected samples on a DNA high-sensitivity chip (Agilent Bioanalyzer), the cDNA concentration from all samples was measured using Quantifluor dsDNA (Promega). Tagmentation was performed in 2 uL volume containing 100 pg amplified cDNA, 1X tagmentation buffer (10 mM Tris-HCl pH 7.5, 5 mM MgCl2, 5% DMF) and 0.1 uL amplicon tagmentation mix (Illumina XT DNA sample preparation kit). After incubation at 55ºC for 10 min, 0.5 uL 0.2% SDS was added to each well to strip off the Tn5 enzyme from the DNA. The tagmented libraries were amplified after addition of 1.5 uL of custom Nextera Index primers (0.5 mM stock; IDT) and 3 uL PCR mix containing 1X Phusion HF buffer (Thermo Scientific), 0.01 U/uL Phusion DNA polymerase (Thermo Scientific) and 0.2 mM dNTP, incubation at 72ºC for 3 min, 98ºC for 3 min, followed by 12 cycles of 98ºC for 10 sec; 55ºC for 30 sec and 72ºC for 30 sec, and a final incubation at 72ºC for 5 min.