title: GSM7074108: s3__4_dpl_; Danio rerio; RNA-Seq
library_construction_protocol: Uninjured and light-lesioned Tg(pcna:EGFP);Tg(gfap:nls-mCherry) fish were exposed to an overdose (0.2%) of MS-222 (Sigma) (Brand et al., 2002) until cessation of the operculum movement. Subsequently, each cornea was poked with a needle, and tweezers were gently pressured on each side of the eye to remove the lens. Then, tweezers were used to carefully isolate the retinae, which were put in a dissociation solution of papain (16 U/ml), dispase (0.2 U/ml) and DNAse (168 U/ml) dissolved in Earle's Balanced Salt Solution (EBSS, Worthington Biochemical Corporation). Up to 6 retinae were put in 1.7 ml of dissociation solution. Retinae were digested for 30 minutes at 28°C, and then calcein blue (final concentration: 1µM, Invitrogen) was added for 10 minutes to the digestion solution at 28°C to stain for living cells. At the end of the enzymatic digestion, mechanical trituration was applied using fire-polished glass pipettes of decreasing tip diameter to help tissue dissociation. Single cells were centrifuged (300 g) at 4°C, for 6 minutes. The cell pellet was re-suspended in EBSS supplemented with DNAse (100 U/ml) to eliminate residual, ambient DNA, and albumin ovomucoid inhibitor (1:10 of stock solution, Worthington Biochemical Corporation) to stop papain action. The single cell solution was eventually filtered (20 µm filters) to remove clumps and debris, and put on ice, ready for Fluorescent Activated Cell Sorting (FACS). Each single cell suspension was obtained by pooling 8 retinae (from 4 fish, 2 males and 2 females) of uninjured and light-lesioned Tg(pcna:EGFP);Tg(gfap:mCherry) animals. Uninjured WT AB, Tg(gfap:mCherry) and Tg(pcna:EGFP) fish were used to prepare single cell suspensions of dissected retinae for setting the single fluorophore gates in flow cytometry. Additionally, a calcein only stained single cell suspension was prepared from uninjured WT AB fish retinae for the setting of the calcein gate in the flow cytometry. Flow cytometry and FACS A BD FACSAria™ III cell sorter was used for flow cytometry and FACS. mCherry fluorophore was detected using a 561 nm excitation laser and a 610/20 nm band pass filter, EGFP using a 488 nm laser and a 530/30 nm band pass filter and calcein blue using a 405 nm laser and a 450/40 nm band pass filter. The gating strategy is shown in Figure 1-supplemental figure 3 for uninjured samples and was the same for all the light-lesioned samples. Forward and side scatters were used to gate cells among all the events in the single cell suspension and to gate singlets among those cells (Figure 1-figure supplement 3A). mCherry-positive only as well as EGFP-positive only gates were set in comparison to the unstained, WT control (Figure1-figure supplement 3B). Calcein blue positive, single cells were eventually gated as living cells (60.000 living cells from uninjured, 44 hpl and 4 dpl samples, 41.389 living cells from 6 dpl sample). 15.000 cells were sorted from each samples, put on ice and sent to the CMCB Deep Sequencing Facility for single cell RNA sequencing (scRNAseq). Single cell transcriptome sequencing was performed based on the 10x Genomics Single cell transcriptome workflow (Zheng et al., 2017). Specifically, 15.000 FACS-sorted cells were recovered in BSA-coated tubes containing 5 ul PBS with 0.04% BSA. Cell samples were carefully mixed with Reverse Transcription Reagent (Chromium Single Cell 3' Library Kit v3) and loaded onto a Chromium Single Cell B Chip to reach a recovery of 10.000 cells per sample.  The samples were processed further following the guidelines of the 10x Genomics user manual for single cell 3' RNA-seq v3. In short, the droplets were directly subjected to reverse transcription, the emulsion was broken and cDNA was purified using silane beads (Chromium Single Cell 3' Gel Bead Kit v2). After amplification of cDNA with 11 cycles, samples were purified with 0.6x volume of SPRI select beads to deplete DNA fragments smaller than 400 base pairs and cDNA quality was monitored using the Agilent FragmentAnalyzer 5200 (NGS Fragment Kit). 10 ul of the resulting cDNA were used to prepare single cell RNA seq libraries - involving fragmentation, dA-Tailing, adapter ligation and 11 cycles of indexing PCR following manufacturer's guidelines. After quantification, both libraries were sequenced on an Illumina NextSeq500 in 75 base pair paired-end mode, generating 45-109 million  fragments per transcriptome library.