title: GSM3167490: cell line B, fish; Danio rerio; RNA-Seq
library_construction_protocol: For inDrop and CEL-Seq samples were obtained using a dissecting forceps and placing the desired tissue sample in 1.5 mL Eppendorf tube followed by addition of 500 uL 0.25% Trypsin-EDTA for digestion. The digestion was carried out at 37ºC in thermomixer for 15-30 min to soften tissue, every 5 minutes mashing the tissue using a disposable pestle to break up softened tissue. Upon completion of incubation at 37ºC, 500 uL of DMEM10 were added to deactivate the trypsin. Cells were washed three times by spinning down the sample at 500 rcf for 5 minutes and resuspended in PBS. Then, sample was filtered twice using 5 mL polystyrene round-bottom tube with 35um cell-strainer. Viability and single cell consistency were checked prior to encapsulation of the cells using the inDrop system for each biopsy taken. For the ST sample sections from zebrafish melanoma tumors were obtained by sectioning the entire tumor with its surrounding. Tissue was gently washed with cold 1X-PBS, and 4-5 mm3 cubes were removed with a scalpel for OCT embedding. Tissue was transferred from 1X PBS to a dry, sterile 10-cm dish and gently dried prior to equilibration in cold OCT for 2 minutes. The tissue was then transferred to a tissue-mold with OCT and snap-frozen in liquid nitrogen-chilled isopentane. Tissue blocks were stored at -80°C until further use. Prior to cryosectioning, the cryostat was cleaned with 100% ethanol, and equilibrated to an internal temperature of -18°C for 30 minutes. Once equilibrated, OCT embedded tissue blocks were mounted onto the chuck and equilibrated to the cryostat temperature for 15-20 minutes prior to trimming. ST slide was also placed inside cryostat to keep the slide cold and minimize RNase activity. Sections were cut at 10 µm sections and mounted onto the ST arrays, and stored at -80°C until use, maximum of two weeks. Prior to fixation and staining, the ST array was removed from the -80C and into a RNase free biosafety hood for 5 minutes to bring to room temperature, followed by warming on a 37°C heat block for 1 minute. Tissue was fixed for 10 minutes with 3.6% formaldehyde in 1X PBS, and subsequently rinsed in 1x PBS. Next, the tissue was dehydrated with isopropanol for 1 minute followed by staining with hematoxylin and eosin. Slides were mounted in 65 µl 80% glycerol and brightfield images were taken on a Leica 397 SCN400 F whole-slide scanner at 40X resolution. For inDrop library construction the cells and reverse transcription (RT) reaction was carried out as previously described in Klein et al., 2015. RNA amplification and library preparation were carried out according to this protocol incorporating the changes introduced in Zilionis et al. 2017. Briefly, RNA was reverse transcribed (RT) with SuperScript III (Invitrogen) in droplets. Droplet emulsions were broken, and post-RT material underwent second strand syntehsis and in vitro transcription using the T7 High Yield Enzyme mix (New England Biolabs). RNA was fragmented for 3 minutes with 1X fragmentation reagent prior to RT with random hexamers, eliminating the need for an adaptor ligation step. Cycles required for final library amplification was assessed by quantitative PCR (qPCR) with KAPA HiFi Hot Start PCR Mix (KAPA Biosystems) and EvaGreen dye (Biotium). Final libraries were amplified with KAPA HiFi Hot Start PCR mix for 9 to 13 cycles. inDrop library size assessed on a DNA BioAnalyzer chip following the manufacturer's instructions (Agilent). For spatial transcriptomics (ST) library construction, we followed the previously published ST protocol from Stahl et al. Science 2016, with minor changes. Briefly, after brightfield imaging of stained tissue, tissue was permeabilized with collagenase and 0.1% pepsin prior to an overnight RT step with SuperScript III on the ST slide. Tissue was digested away with proteinase K and 1% 2-mercaptoethanol prior to cleavage of probes from the slide surface with USER enzyme. Second strand synthesis was performed with DNA Pol I and RNase H (New england biolabs) followed by an in vitro transcription amplification step with the MEGAScript T7 kit (Thermofisher). Amplified RNA then underwent a second RT with random hexamers and SuperScript II (Invitrogen), eliminating the need for an adaptor ligation step. Cycles required for final library amplification was assessed by quantitative PCR (qPCR) with KAPA HiFi Hot Start PCR Mix (KAPA Biosystems) and EvaGreen dye (Biotium). Final libraries were amplified with KAPA HiFi Hot Start PCR mix for 9 to 13 cycles. inDrop library size assessed on a DNA BioAnalyzer chip following the manufacturer's instructions (Agilent). For CEL-Seq2 sample, cells were sorted using fluorescence activated cell sorting (FACS) into 384 well plate contacting 1.2ul primer mix and library was constructed according to the CEL-Seq2 protocol (Hashimshony et al. 2016).
attributes.tag: GEO Accession
attributes.value: GSM3167490