GSM4564024: mouse embryonic stem cells - day 1 - column 1 - rows 1:40 RapidRun; Mus musculus; RNA-Seq
Cells were rinsed thoroughly with PBS, trypsinized for 7 minutes and resuspended in the respective growth medium at a concentration of 400 cells/μl. 30 μl cell suspension was diluted with 20 μl of suspension reagent (Fluidigm) and 10 μl of the dilution was loaded into one compartment of a Single-Cell mRNA Seq HT integrated fluidic circuit (IFC) 10-17 µm. A different cell type was loaded into the other compartment, which is not analyzed in this study. Cell viability staining was performed on the IFC using the LIVE/DEAD viability/Cytotoxicity Kit (Thermofisher) with 1 μM Ethidium and 0.05 μM Calcein. IFC loading and life/dead staining was analyzed with automated image acquisition using a Zeiss CellDiscoverer microscope (Zeiss) with a 20x objective. During the lysis step ERCC Spike-in Mix 1 (Thermofisher) was added with a final dilution of 1:200000. Lysis, reverse transcription and cDNA amplification was performed on the C1 machine. Single-cell RNA-seq libraries were prepared with the C1-HT mRNA-seq v2 protocol according to the manufacturer's recommendations (Fluidigm). cDNA pools were quantified by Qubit and Bioanalyzer HS. Around 2.25 ng of each pool were subjected to tagmentation and library preparation using the NexteraXT library preparation kit according to the C1-HT protocol. All pools were mixed in equal proportions and quantified with KAPA Library Quant-Kit. The libraries were sequenced on a HiSeq2500 instrument (Illumina) either in high output or in rapidrun mode, in asymmetric read length (Read1: 13bp, Index read: 8pb, Read2: 48bp), with 10pM loading concentration and 5% PhiX.