title: GSM8839424: control_WT; Mus musculus; RNA-Seq
library_construction_protocol: Mice were euthanized by CO2 exposure followed by cardiac perfusion with 10 ml ice-cold PBS. Eyes were immediately dissected to isolate the retinae. Retinae were dissected in 500µl 0.25mM DMEM/HEPES containing 120 U/ml papain (Carl Roth, #9001-73-4), 225 U/ml collagenase IV (Worthington, #LS004188) and 20 U/ml DNase I (Worthington, #LS006344). Prior to digestion at 8°C for 45 minutes, each sample was mechanically triturated by pipetting. After digestion, cell suspensions were filtered through a 50µm cell strainer. Retinae from 3 to 5 mice were pooled to obtain sufficient cell numbers. To  remove dead cells and debris, cell suspension was incubated with magnetic beads (DeadCell Removal Kit, Milteny, # 130-090-101). Cells were sorted through MS Columns (Milteny, # 130-042-201) installed in an OctoMacs magnet (Milteny, # 130-042-109) according to manufactures protocol. Intact and viable single cells were collected and resuspended in ultra-pure BSA (1:1000, Thermo Fisher, #AM2616). Prior to processing for sc RNAseq, the single-cell suspensions were again filtered through a 40 µm strainer. Cell concentrations were assessed using an automated cell counter. The concentration for each sample was adjusted to approximately 1,000 cells/µL resulting in approx. 10,000 cells per reaction. We used the 3' 10x Genomics Chromium Single Cell Gene Expression protocol (v3.1) to capture and barcode mRNA transcripts from the single cell suspensions.  Briefly, the cell suspensions, together with barcoded gel beads, master mix, and partitioning oil, was loaded on a 10x Genomics Chromium Next GEM chip. Using a Chromium Controller, the cells were partitioned into "gel bead in emulsions" (GEMs). Within each GEM, reverse transcription was followed by barcoding of transcripts with a unique 10x barcode and unique molecular identifiers (UMIs). The cDNA was purified using Silane dynabeads followed by PCR amplification. The amplified cDNA was fragmented, end repaired and A-tailed to create compatible ends for adapter ligation. Sequencing adapters (P5 and P7) were ligated to the cDNA fragments, and a sample index was added during a final round of PCR amplification, enabling multiplexing of samples in a single sequencing run. The constructed libraries were purified using AMPure XP beads before cDNA concentration and fragment size distribution were assessed using a Qubit Fluorometer and an Agilent Bioanalyzer. Libraries were pooled in equimolar ratios for sequencing. Libraries were sequenced on an Illumina NovaSeq 6000 platform using the 100 cycle S2 reagent kit in paired-end mode. Sequencing parameters were set to 28 bp for Read 1 (cell barcode and UMI), 8 bp for the i7 index (sample index), and 91 bp for Read 2 (cDNA insert). We aimed for a sequencing depth of approximately 50,000 reads per cell.
identifiers.namespace: GEO
identifiers.id: GSM8839424_r1