title: Illumina NovaSeq 6000 paired end sequencing; Single-cell RNA sequencing of germ-free zebrafish embryos
design: Single-cell RNA sequencing of germ-free zebrafish embryos
library_construction_protocol: The generation of germ-free zebrafish (wild-type AB) followed previously established protocols [1]. In brief, 2 hpf embryos were transferred to petri dishes with sterile E3 medium, supplemented with ampicillin (100 μg/mL), kanamycin (5 μg/mL) and amphotericin B (250 ng/mL) and incubated at 28°C. At 50% epiboly up to shield stage (6 hpf), the embryos were surface disinfected with 0.1% of polyvinylpyrroidone-iodine (PVP-I) for exactly 2 min, followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium, transferred to flasks and incubated at 28°C. The viability was monitored, and sterile medium was refreshed daily. At day 4, the hatched embryos were used for the sterility validation. A day 5, germ-free zebrafish were exposed to the combination of GO (30 µg/mL) and butyrate (2.5 mM) for 24 h. GO and BA were pre-incubated for 1 h prior to the exposure. Twenty larvae were used as one replicate, four replicates, i.e. eighty larvae in total, were used for each condition. After the exposure, zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly, zebrafish larvae were euthanized with 0.01% of tricaine for 5 min, collected in 1.5 mL tube, and washed with PBS for three times. The dissociation was initiated by adding 500 μL of pre-warmed enzyme mix containing 460 μL of 0.25% trypsin-EDTA (Gibco) and 40 μL of collagenase (Sigma-Aldrich) at the concentration of 100 mg/mL, followed by mechanical dissociation using P1000 and then P200 pipette tips in heat-block at 30 °C until the tissue was no longer visible (about 10 min). The dissociation was then stopped by adding 800 μL DMEM (Corning Cat# 10-013-CV) with 10% FBS (Fisher Scientific). The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT, followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 μm nylon mesh, with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non-viable cells (Invitrogen Cat# D15106) at the dose of 3 μM for 10 min at room temperature before the fluorescence-activated cell sorting (BD FACSAria III, BD Biosciences, NJ, USA). The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 °C and gated based on forward scatter and DRAQ7. The DRAQ7 negative cells of each sample were FACS sorted into the tubes containing DMEM with 10% FBS and placed on ice immediately after the sorting. The cells were then proceeded with the single-cell RNA sequencing analysis. 1. Pham LN, Kanther M, Semova I & Rawls JF. Methods for generating and colonizing gnotobiotic zebrafish. Nat. Protoc. 3, 1862-1875 (2008). 2. Bresciani E, Broadbridge E, Liu PP An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae, MethodsX. 5, 1287-1290 (2018). Single-cell RNA sequencing (scRNA-seq) was performed by Eukaryotic Single Cell Genomics Facility at SciLifeLab, Stockholm. The single cells collected from FACS sorting were loaded on a 10x GemCode Single-Cell Instrument (10x Genomics, Pleasanton, USA) to generate single-cell gel beads in emulsion (GEMs) using the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1 (10x Genomics) following the manufacture's protocol.  Briefly, GEMs were generated by combining barcoded Single Cell 3' v3.1 Gel Beads, a Master Mix containing cells, and Partitioning Oil onto Chromium Next GEM Chip G. Following GEMs generation, the Gel Bead was dissolved, primers containing an Illumina TruSeq Read 1(read 1 sequencing primer), 16 nt 10x Barcode, 12 nt unique molecular identifier (UMI) and 30 nt poly(dT) sequence were released, and the co-partitioned cells were lysed.  The nucleic acid library construction was performed using the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1 (10x Genomics, 16 rxns PN-1000268) following the manufacture's protocol. After GEMs generation, primers were mixed with the cell lysate and a Master Mix containing reverse transcription (RT) reagents to produce barcoded, full-length cDNA from polyadenylated mRNA. Silane magnetic beads were used to purify the first-strand cDNA from the post GEM-RT reaction mixture, which included leftover biochemical reagents and primers. Barcoded, full-length cDNA was amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. P5, P7, i7 and i5 sample indexes, and TruSeq Read 2 (read 2 primer sequence) were added via End Repair, A-tailing, Adaptor Ligation, and PCR. The final libraries containing the P5 and P7 primers used in Illumina amplification were prepared for an estimated 5000 nuclei per sample.