run_metadata: 48408

This data as json

rowid	run.accession	experiment.accession	sample.accession	study.accession	bioproject	study.title	study.alias	study.type	study.abstract	study.attributes	study.PMIDs	sample.description	sample.title	sample.alias	sample.centername	sample.attributes	GEOsample.title	GEOsample.dataprocessing	GEOsample.source	GEOsample.treatmentprotocol	GEOsample.extractprotocol	GEOsample.growthprotocol	GEOsample.characteristics	GEOsample.accession	experiment.title	experiment.alias	experiment.library_name	experiment.design_description	experiment.library_construction_protocol	experiment.attributes	experiment.library_strategy	experiment.library_source	experiment.library_selection	experiment.library_layout	experiment.platform	experiment.instrument_model	experiment.spot_descriptor	experiment.study_ref	run.title	run.attributes	run.filename	run.semantic_name	run.total_bases	run.total_spots	run.alias	run.read_lengths	run.base_counts	run.r1_length	run.r2_length	run.r3_length	run.r4_length	run.Acount	run.Ccount	run.Gcount	run.Tcount	run.Ncount	run.experiment	run.pool_member	submission.accession	submission.srasource	submission.bioprojectsource	seqdetective.n_mates	seqdetective.mapping_rate.mate1	seqdetective.mapping_rate.mate2	seqdetective.nofeature_rate.mate1	seqdetective.nofeature_rate.mate2	seqdetective.sparsity.mate1	seqdetective.sparsity.mate2	seqdetective.pos_strand_rate.mate1	seqdetective.pos_strand_rate.mate2	seqdetective.readlen.mate1	seqdetective.readlen.mate2	seqdetective.judgement.mate1	seqdetective.judgement.mate2	seqdetective.judgement.reason	platform_family	instrument_generation	read_bias	selection_class	prep_kit	sc_or_bulk	tech_class	technology	tech_variant	submission.bioprojectsource.country	earliest_date	devstage_curation	devstage_curation_coarse	tissue_curation	tissue_curation_coarse
48408	SRR7240611	SRX4146442	SRS3360062	SRP149420	PRJNA473915	Single cell analysis of tumor progression reveals the function structure and evolution of cancer archetypes	GSE115140	Transcriptome Analysis	The classic cancer evolution model posits that driver mutations sweep the population sequentially as the complete set of hallmarks are assembled by the neoplastic clone. However recent work has challenged this model revealing that most tumors contain highly complex dynamics with genetic diversity reflecting distinct clonal architectures. The functional and phenotypic heterogeneity also has been shown to have a crucial influence on the fate of the tumor5–10. However it is not well understood how distinct tumor clonal populations coexist and function. Here we study tumor architecture at the level of individual cells by sampling a zebrafish melanoma tumor over time and space. We found that cancer transcriptional programs can be classified to three archetypes each exploiting the existing neural crest mature melanocytes and stress modules and distinct intra tumor locations. Strikingly these archetypes are conserved in human melanoma. Further we found that the cancer cells are comprised of two distinct clones where one expresses a unique archetype. Over time we found that the cells of this clone adapt by exhibiting a more similar profile to the corresponding archetype. Overall design: Single cell RNA sequencing of zebrafish tumor cells from 2 zebrafish at multiple time points.				ZF2 time point 1	GSM3167484		tissue:tumor cells\|time:2017 05 25\|type:inDrop	ZF2 time point 1	Illumina RTA v2 software was used for basecalling and quality determination. Raw sequencing data obtained from the inDrop method was processed using a custom built pipeline available at https://github.com/flo compbio/singlecell. Briefly the location of the known “W1” adapter sequence of the inDrop RT primer was located in the barcode read read 2. Reads for which the W1 sequence could not be detected were discarded. The start position of the W1 sequence was then used to infer the length of the first part of the inDrop cell barcode in each read which can range from 8 11 bp as well as the start position of the second part of the inDrop cell barcode which is 8 bp long. Cell barcode sequences were mapped to the known list of 384 barcode sequences for each read. The resulting barcode combination was used to identify the cell from which the fragment originated. Finally UMI sequence was extracted and reads with low confidence base calls for the six bases comprising the UMI sequence minimum PHRED score less than 20 were discarded. The reads containing the mRNA sequence read 1 were mapped using STAR with parameter “—outSAMmultNmax 1” and default settings otherwise27. Expression was quantified by counting the number of reads mapped to each gene and correcting for UMI as described previously Grün et al. 2014 The genome and gff file used included the zebrafish genome and the BRAF human vector. Single cell transcriptomes with UMIs>750 mitochondrial transcripts < 20% and ribosomal transcripts < 30% were retained for analysis. Raw sequencing data obtained from the Spatial Transcriptomics ST method were processed using a publicly available pipeline https://github.com/jfnavarro/st pipeline. Briefly quality trimming is performed to remove low quality bases and reads with long nucleotide stretches > 15. Read 2 transcript sequence is mapped with STAR 2.5.1 and Read 1 spatial barcode is demultiplexed with Taggd. Reads that contain both a valid spatial barcode and are correctly map are kept. UMIs are then counted with htseq count; to get a final read count annotated reads are grouped by spatial barcode. Raw sequencing data obtained from the CEL Seq2 method were processed using a publicly available pipeline https://github.com/yanailab/celseq2. Genome build: Danio rerio.GRCz10 Supplementary files format and content: For inDrop data: tab delimited files with genes as rows and cells as columns. For ST data: tab delimited files with spots x and y coordinates for the ST array as columns and genes as rows. For CEL Seq data: csv files with genes as rows and cells as columns.	tumor cells		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 post 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.		time:2017 05 25\|type:inDrop	GSM3167484	GSM3167484: ZF2 time point 1; Danio rerio; RNA Seq	GSM3167484		1	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 post 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.	GEO Accession:GSM3167484	RNA-Seq	TRANSCRIPTOMIC	cDNA	PAIRED	ILLUMINA	NextSeq 500		SRP149420			BS00417A_S1_R1_001.fastq.gz BS00417A_S1_R2_001.fastq.gz	fastq fastq	15195762260.0	176694910.0	GSM3167484 r1	0:35 1:51	A:3730288221;C:3100069649;G:3917397047;T:4447806244;N:201099	35	51			3730288221	3100069649	3917397047	4447806244	201099	SRX4146442	SRS3360062	SRA713129	GEO	Yanai, NYU	2	0.45584	0.02194	0.10866	0.01434	0.8508	0.99594	0.56562	0.74387	35	51	B	T	mate2 technical by mapping diff	illumina	nextseq	unknown	random_priming	unknown	sc	single_cell_droplet	indrops		United States	2018-05-31	Undetermined	Undetermined	Cancer or Tumor	Cancer or Tumor