title: GSM2975749: C57Bl/6 mouse whole bone marrow cells; Mus musculus; RNA-Seq
library_construction_protocol: Whole bone marrow cells were isolated from 11 weeks old C57Bl/6 male mice (Jackson Laboratory). Epiphysis/metaphysis fraction from long bones was collected, crushed, cut into small pieces, and digested using Collagenase I (STEMCELL Technologies) for 30 minutes at 37-degree agitation.  Bone marrow cells were filtered through a 70-micron filter. Red blood cells were lysed using Ack-lysis (ThermoFisher Scientific) on ice for five min, quenched with Media 199 (ThermoFisher Scientific) supplemented with 2% fetal bovine serum (ThermoFisher Scientific), and spun down at 500 g for 5 min. Cells were stained for 30 minutes with the red blood cell marker TER119 (Biolegend) and cells were sorted using DAPI (ThermoFisher Scientific) as a live/dead viability marker. Four hundred thousand live whole bone marrow cells (negative for TER119) were sorted into medium 199 (ThermoFisher Scientific). Before inDrop encapsulation cells were counted using a Cellometer (Nexcelom Bioscience). Cell viability is over 90%. InDrop processing. Concentration of cells was adjusted to 300,000 cells/ml by adding PBS to the sorted cells. The cell suspension was then mixed 1:1 (v/v) with PBS containing 30% OptiPrep Density Gradient Medium (Sigma D1556) to obtain 150,000 cells/ml in 15% Optiprep. Using four microfluidics pumps and a polydimethylsiloxane (PDMS) microfluidic device, about 10,000 cells were co-encapsulated with barcoded polyacrylamide beads (V3) and a reverse transcription mixture containing Superscript III into water-in-oil droplets, according to a previously published protocol. The library preparation and quality control procedures were carried out as described previously. Indexed libraries were pooled and sequenced on a Next-seq 500 system (Illumina) at 2 pM concentrations. single-cell RNA-seq (inDrop V3)
attributes.tag: GEO Accession
attributes.value: GSM2975749