GSM7389053: CAAX control, 24hpi, biological replicate 2; Danio rerio; RNA-Seq
Whole larvae were dissociated by collagenase and rapid pipetting. Dissociation Solution consisted of HBSS (no mag, no cal, plus phenol red), HEPES 15mM , D-Glucose 25mM, 2% Sterile Goat Serum, Collagenase IV 2.5mg/ml. Groups of 50 larvae were submerged in 2ml of Dissociation Solution prewarmed to 28 degrees and transferred to a 12 well plate. Each well was incubated at 28 degrees with shaking for 5 minutes, followed by 1 minute of rapid pipetting through a P1000 filter tip. Incubation and pipetting were repeated a further two times until a single cell suspension was obtained. For each sample, 5 wells were pooled into a single falcon tube, then washed and resuspended in 500ul of Dissociation Solution sans collagenase. Samples were filtered and stained with DAPI shortly prior to sorting using a FACS Fusion (Becton Dickson). Keratinocytes/preneoplastic cells and myeloid cells were sorted into a single tube for each sample on the basis of mcherry and EGFP expression respectively. Freshly collected cells were washed once in HBSS at 4 degrees. Cells were counted and checked for viability with a cytometer and approx. 7000 cells were loaded to the 10X chromium chip. Library preparation was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, cells were resuspended in master mix and loaded onto the 10X chip alongside partitioning oil and beads to form GEMs (gel beads in emulsion). A poly-A capture method was used to reverse transcribe each transcript within each GEM whilst adding a Unique Molecular Identifier (UMI), cell barcode and Illumina R1 primer sequence. The resulting cDNA was captured on Silane DynaBeads and washed prior to amplification by PCR. To prepare the libraries, further rounds of PCR were carried out, in which the Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.