Illumina HiSeq 2000 paired end sequencing; Single-cell RNA sequencing of spleen-derived LCK-expressing cells from adult zebrafish
Single-cell RNA sequencing of spleen-derived LCK-expressing cells from adult zebrafish
Spleens from two heterozygote Tg(lck:EGFP) fish were dissected and passed through a 40μm cell strainer using the plunger of a 1-mL syringe and cells were collected in cold 1xPBS/5% FBS. A non-transgenic line was used to set up the gating and exclude autofluorescent cells. Propidium iodide (PI) staining was used to exclude dead cells. Individual cells were sorted, using a Becton Dickinson Influx sorter with 488- and 561 nm lasers (Schulte et al., 2015) and collected in a single well of a 96 well plate containing 2.3 uL of 0.2 % Triton X-100 supplemented with 1 U/uL SUPERase In RNAse inhibitor (Ambion). The size, granularity and level of fluorescence for each cell were simultaneously recorded.  The Smart-seq2 protocol (Picelli et al., 2014) was used to amplify the whole transcriptome and prepare libraries. Twenty-five cycles of PCR amplification were performed.