title: GSM4828393: Megakaryocyte Progenitor (MkP) 126; Mus musculus; RNA-Seq
library_construction_protocol: Whole bone marrow cells were isolated by crushing long bones with mortar and pestle using PBS/2% FCS/2 mM EDTA and filtered through a 100 µm mesh. After erythrocyte lysis in hypotonic NH4Cl-buffer, cells were filtered through a 40 µm mesh. Hematopoietic lineage+ cells were removed with the lineage cell depletion kit (Miltenyi Biotec). Cells were incubated with antibodies in PBS / 2% FCS for 30 min, washed twice with PBS / 2% FCS. Single BM HSPC cells were sorted on an BD ARIA II SORP flow cytometer. Single hematopoietic stem and progenitor cells were index-sorted into 384 well plates containing 0.5 µl of nuclease free water with 0.2% Triton-X 100 and 4 U murine RNase Inhibitor (NEB), spun down and frozen at -80°C. After thawing, 0.5 µl of a primer mix were added (5 mM dNTP (Invitrogen), 0.5 µM dT-primer (C6 aminolinkerAAGCAGTGGTATCAACGCAGAGTCGACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN), 1 U RNase Inhibitor (NEB)). RNA was denatured for 3 minutes at 72°C and the reverse transcription (RT) was performed at 42°C for 90 min after filling up to 10 µl with RT buffer mix for a final concentration of 1x superscript II buffer (Invitrogen), 1 M betaine, 5 mM DTT, 6 mM MgCl2, 1 µM TSO-primer (AAGCAGTGGTATCAACGCAGAGTACATrGrGrG), 9 U RNase Inhibitor and 90 U Superscript II. After synthesis, the reverse transcriptase was inactivated at 70°C for 15 min. The cDNA was amplified using Kapa HiFi HotStart Readymix (Peqlab) at a final 1x concentration and 0.1 µM UP-primer (AAGCAGTGGTATCAACGCAGAGT) under following cycling conditions: initial denaturation at 98°C for 3 min, 23 cycles [98°C 20 sec, 67°C 15 sec, 72°C 6 min] and final elongation at 72°C for 5 min. The amplified cDNA was purified using 1x volume of hydrophobic Sera-Mag SpeedBeads (GE Healthcare) resuspended in a buffer consisting of 10 mM Tris, 20 mM EDTA, 18.5 % (w/v) PEG 8000 and 2 M sodium chloride solution. The cDNA was eluted in 12 µl nuclease free water and the concentration of the samples was measured with a Tecan plate reader Infinite 200 pro in 384 well black flat bottom low volume plates (Corning) using AccuBlue Broad range chemistry (Biotium). For library preparation up to 700 pg cDNA was desiccated and rehydrated in 1 µl Tagmentation mix (1x TruePrep Tagment Buffer L, 0.1 µl TruePrep Tagment Enzyme V50; from TruePrep DNA Library Prep Kit V2 for Illumina; Vazyme) and tagmented at 55°C for 5 min. Subsequently, Illumina indices are added during PCR (72°C 3 min, 98°C 30 sec, 13 cycles [98°C 10 sec, 63°C 20 sec, 72°C 1 min], 72°C 5 min) with 1x concentrated KAPA Hifi HotStart Ready Mix and 300 nM dual indexing primers. After PCR, libraries are quantified with AccuBlue Broad range chemistry, equimolarly pooled and purified twice with 1x volume Sera-Mag SpeedBeads. This was followed by Illumina 50 bp paired-end sequencing on a Novaseq6000 aiming at an average sequencing depth of 0.5 mio reads per cell.