title: GSM5830169: scRNAseq_RANKL_D3_1; Mus musculus; RNA-Seq
library_construction_protocol: After harvesting, organoids were dissociated using TrypLE (Gibco). Viable single cells were sorted in 384-well PCR plates (Bio-Rad) using the Becton Dickinson Aria (BD Biosciences) sorter with a 100 µm nozzle, based on forward and side scatter gating. Prior to sorting, plates were primed with oligos (5'-3') containing the T7 promoter, a 5' Illumina adapter, unique molecular identifiers (UMIs), a unique cell barcode and the oligo-(dT)N for tagging of individual mRNA molecules within each cell (Gerlach et al,. 2019). Immediately after sorting, plates were centrifuged for 2 minutes at 1.200 x g and 4°C and stored at -80°C until library preparation. Libraries were prepared using a modified CEL-Seq2 protocol (Hashimshony et al., 2016). In brief, a micro-dispenser machine Nanodrop II (BioNex) was used to dispend in each well the ERCC spike-in control RNAs (1:50.000, Invitrogen), reagents used for the reverse transcription (RT) reaction such as Superscript II (Invitrogen) and RNasein Plus (Promega), and for the second strand synthesis such as E. coli DNA polymerase and E. coli DNA ligase (New England Biolabs). Second strand synthesis was done in the presence of E. coli RNase H (Invitrogen). Double stranded cDNA from each well was pooled together and purified with AMPure XP beads (Beckman Coulter). In vitro transcription was performed overnight according to the Ambion MegaScript IVT kit protocol (Invitrogen), followed by an exonuclease digestion step with EXOI/rSAP-IT (Applied Biosystems). The amplified RNA was later fragmented and cleaned up using AMPure XP beads (Beckman Coulter). Samples were then subjected to library RT using random octamers (Gerlach et al,. 2019) and to cDNA amplification thereafter using the Phusion High-Fidelity DNA Polymerase (New England Biolabs). A forward primer sequence against the 5' Illumina adapter (Gerlach et al,. 2019) and a reverse primer sequence against the octamers overhang incorporated during RT were added to the amplification mix. The reverse primer contained an Illumina 3' adapter sequence and an index sequence to uniquely identify each library (NextFlex DNA barcodes, PerkinElmer). Libraries were purified and quantified using the dsDNA Fluorescence Quantification Assays (DeNovix) and fragment sizes were assessed with the BioAnalyzer High Sensitivity DNA Kit (Agilent Technologies). The libraries were sequenced on an Illumina NextSeq 500, and 63-bp paired-end reads were generated.