GSM6005157: copMG_2; Homo sapiens; RNA-Seq
GSM6005157
Co-cultures were first dissociated by 15 min incubation with papain (P4762, Sigma-Aldrich) diluted in accutase (20 U/mL) and gentle trituration based on a previously published protocol (Thiry et al., 2020). The cell suspension was then passed through a cell strainer (70 μm, Falcon) to remove cell clumps. To extract co-pMG, magnetic-activated cell sorting (MACS) was then performed using CD11b-MACS beads (130-093-634, Miltenyi Biotec) according to the manufacturer's instructions. The panned cell population was lysed using an RNAeasy Micro kit (Qiagen) according to the manufacturer's instructions Material was quantified using RiboGreen (Invitrogen) on the FLUOstar OPTIMA plate reader (BMG Labtech) and the size profile and integrity analysed on the 2200 or 4200 TapeStation (Agilent, RNA ScreenTape). RIN estimates for all samples were between 9.2 and 9.9. Input material was normalised to 100 ng prior to library preparation. Polyadenylated transcript enrichment and strand specific library preparation was completed using NEBNext Ultra II mRNA kit (NEB) following manufacturer's instructions. Libraries were amplified (14 cycles) on a Tetrad (Bio-Rad) using in-house unique dual indexing primers (based on (Lamble et al., 2013)). Individual libraries were normalised using Qubit, and the size profile was analysed on the 2200 or 4200 TapeStation. Individual libraries were normalised and pooled together accordingly. The pooled library was diluted to ~10 nM for storage. The 10 nM library was denatured and further diluted prior to loading on the sequencer