alias: GSM4544441
title: GSM4544441: mouse LSK, LCE410; Mus musculus; RNA-Seq
library_construction_protocol: marrow cells from either C57BL/6 mice or Dach1-GFP mice were harvested, made into a single cell suspension, incubated with red cell removal buffer (NH4Cl), followed by incubation with anti-CD117-APC, then incubation with anti-APC magnetic beads, followed by purification on a LS column (Miltenyit biotech). This enriched fraction was then stained with a cocktail of antibodies including anti-Sca1, anti-Il7Ra-biotin, anti-CD16/32, anti-CD135-PE, anti-CD150, anti-CD48. Cells were sorted either in bulk or using the single cell mode of a BD Fusion cell sorter. 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.
identifiers.namespace: GEO
identifiers.id: GSM4544441