title: GSM6458051: APHSpleen_WT_Ter119_RNASEQ_3; Mus musculus; RNA-Seq
library_name: GSM6458051
library_construction_protocol: Cells were washed with cold PBS/10% FBS and resuspended in 10 μl of cold PBS/10% FBS per 106 cells and stained with a 1/100 dilution of anti-Ter119-PE antibody (BD Pharmingen 553673 2 μg/ml) at 4 °C for 20 minutes. Stained cells were washed with cold PBS/10% FBS and resuspended in 8 μl of cold PBS/0.5% BSA/2 mM EDTA and 2 μl of anti-PE MACS microbeads (Miltenyi Biotec: 130-048-801) per 106 cells and incubated at 4 °C for 15 minutes. Ter119+ cells were positively selected via MACS lineage selection (LS) columns (Miltenyi Biotec: 130-042-401) and processed for downstream applications. Purity of the isolated erythroid cells was routinely verified by Fluorescence-Activated Cell Sorting (FACS). Total RNA was isolated from 105-2x106 Ter119+ erythroid cells lysed in TRI reagent (Sigma-Aldrich: T9424) using a Direct-zol RNA MiniPrep kit (Zymo Research: R2050). DNase I treatment was performed on the column as recommended in the manufacturer's instructions but with an increased incubation of 30 min at room temperature (rather than 15 min). For RNA-seq libraries, 1-2 μg of total RNA was depleted of rRNA and globin mRNA using the Globin-Zero Gold rRNA Removal Kit (Illumina: GZG1224) according to the manufacturer's instructions. To enrich for mRNA, poly(A)+ RNA was isolated, strand-specific cDNA was synthesised, and the resulting libraries prepared for Illumina sequencing using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs: E7490) and the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs: E7760) following the manufacturer's instructions. Poly(A)+ and Poly(A)- RNA-seq libraries were sequenced on the Illumina Nextseq platform using a 75-cycle paired-end kit (NextSeq 500/550 High Output Kit v2.5: 20024906).