title: GSM4649099: Mouse LSK [RPI4]; Mus musculus; RNA-Seq
library_construction_protocol: Bone marrow from C57BL/6-TG (UBC-GFP) was extracted and cells enriched for ckit (2B8 conjugated to APC) via magnetic activated cell sorting (MACS) positive selection using anti-APC beads (Miltenyi Biotec). The ckit-enriched fraction was subsequently stained for Sca-1 (E13 161-7 conjugated to Alexa Fluor 594 or Alexa Fluor 680), IL-7R (A7R34-2.2 conjugated to biotin with streptavidin-PECy7). C-kithi Sca1+IL-7R¬–CD11blo HSPCs were sorted using the single cell mode of a BD Influx, BD FacsARIA W or BD FacsAria L. Single cells, or 100-cell population controls, were sorted into 96 well round bottomed wells pre-seeded with 1.5 x 106 total C57BL/6 BM cells (filler cells) and cultured in Flt3L conditioned medium at 37 °C and 5% CO2 for 2.5 days. For CEL-seq2, single cells were flow sorted into chilled 384-well PCR plates containing 1.2ul of primer/ERCC mix using a BD FACSAria III flow cytometer. Sorted plates were sealed and immediately frozen upside down at -80C. Then these plates, together with the mRNA mixture plates, were taken from -80C and processed using an adapted Cel-Seq2 protocol with the following variations. We pooled the samples after the first strand synthesis and the second strand synthesis was performed using NEBNext Second Strand Synthesis module. NucleoMag NGS Clean-up and Size select magnetic beads were used for all DNA purification and size selection steps.
attributes.tag: GEO Accession
attributes.value: GSM4649099