title: Illumina NovaSeq 6000 sequencing; Single-cell RNA sequencing of mouse Naïve State and Epiblast Stem Cell like cells
design: Single-cell RNA sequencing of mouse Naïve State and Epiblast Stem Cell like cells
library_construction_protocol: Cell line generation Individual mouse Embryonic Stem Cell lines were derived from different blastocysts of well-defined inbred strains and genetically diverse outbred mice (Diversity Outbred) derived from the Collaborative Cross (“The Collaborative Cross, a community resource for the genetic analysis of complex traits,” 2004).   Cell line maintenance and differentiation mESC lines were thawed on CF1 irradiated mouse embryonic fibroblasts in KSR medium supplemented with 10ng/mL LIF.  Cells were transferred and grown in 2iL condition (N2B27 supplemented with 10ng/mL LIF, 3μM CHIR99021 and 1μM PD0325901), they were passaged every two days using TrypLE.  For differentiation towards EpiSCL cells the supplements were removed for the indicated number of days.  Conversion was performed following the 10X protocol (https://www.10xgenomics.com/) Chromium 10X platform. Library was constructed according to User Guide for Chromium Single Cell 3ʹ Reagent Kits v3 - CG000183 Rev A (https://assets.ctfassets.net/an68im79xiti/4tjk4KvXzTWgTs8f3tvUjq/2259891d68c53693e753e1b45e42de2d/CG000183_ChromiumSingleCell3__v3_UG_Rev_C.pdf [assets.ctfassets.net]) cDNA was always run on Tapestation 4200 with High Sensitivity D5000 assay while final libraries with High Sensitivity D1000 assay - however in few occasions Bioanalyser 2100 with High Sensitivity Kit was used for both cDNA and final libraries. After that QC final libraries were qPCR using QuantStudio 6 and Kapa Biosystems ROX LOW QPCR MASTER MIX. Basing on that quantification libraries were normalised to 15nM and pooled into SLX tubes.