GSM9487363: SPN d21 Sample 1; Mus musculus; RNA-Seq
One hundred ng of total RNA per sample were utilized as input for mRNA enrichment procedure with 'NEBNext® Poly(A) mRNA Magnetic Isolation Module' (E7490L; New England Biolabs, NEB) followed by stranded cDNA library generation using 'NEBNext® Ultra II Directional RNA Library Prep Kit for Illumina' (E7760L; NEB), according to the user manual E7760 (Version 1.0_02-2017) except that all reactions were downscaled to 2/3 of initial volumes. One additional purification step was introduced at the end of the standard procedure, using 1x 'Agencourt® AMPure® XP Beads' (#A63881; Beckman Coulter). Library preparation for single cell mRNA-Seq analysis was performed according to the Chromium NextGEM Single Cell 3ʹ Reagent Kits v3.1 User Guide (Manual Part Number CG000204 Rev B; 10x Genomics). A twofold excess of cells was loaded to the 10x controller in the specified volume in order to reach a target number of 10,000 cells per sample. Fragment length distribution of generated cDNAs and libraries was monitored using 'Bioanalyzer High Sensitivity DNA Assay' (5067-4626; Agilent Technologies). Quantification of libraries was performed by use of the 'Qubit® dsDNA HS Assay Kit' (Q32854; ThermoFisher Scientific).