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| Status |
Public on Jun 04, 2021 |
| Title |
HYR__3ac717__20201006_non-exo_S2 |
| Sample type |
SRA |
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| Source name |
50:50 mixture of human MM087 cells and mouse melanoma cells
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| Organisms |
Homo sapiens; Mus musculus |
| Characteristics |
tissue/cell line: Human: melanoma cell line/mouse: melanoma cell line
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| Growth protocol |
MM087 mouse melanoma cells were cultured in F-10 Nutrient mix supplemented with 10% FBS and 1% penicillin/streptomycin and passaged once per week. Mouse melanoma cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin and passaged once per week.
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| Extracted molecule |
cytoplasmic RNA |
| Extraction protocol |
NOT PROVIDED; REQUESTED
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 2000 |
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| Data processing |
For HyDrop-ATAC samples (prefix HYA):
Barcode reads (read 2) were trimmed to exclude the intersub-barcode PCR adapters MAWK
Barcode reads (read 2) were trimmed using trimgalore (trim_galore -j 2 -o . $R1 $R2 --paired --gzip), corrected to a whitelist allowing 1 mismatch (custom seq script inVSN pipeline version 41df185308 [develop_atac]), and appended to the fastq sequence identifier of the paired-end ATAC-seq reads (read 1 and read 3)
Cell barcodes were written to the bam as a CB SAM tag using SAMtools and AWK (custom VSN pipeline version 41df185308 [develop_atac] script) (samtools view -h HYA__combined__20210323_cortex_phu_dv_etssb_1-5_S5.bwa.out.possorted.bam | mawk '/^@/ {print;next} {N=split($1,n,"_"); NN=split(n[1],nbc,"-"); ucorr_bc=""; corr_bc=""; if(NN==1){ # BioRad data with format {corrected_bc}_qname; corr_bc="CB:Z:" nbc[1];} if(NN==2){ucorr_bc="CR:Z:" nbc[1]; if(length(nbc[2])>0){corr_bc="
Reads were mapped to reference genomes using bwa-mem and duplicate-marked using samtools markdup by the VSN pipeline version 41df185308 [develop_atac] (bwa mem -t 8 genome.fa $R1.fq.gz $R2.fq.gz | samtools fixmate -@ 6 -m -u -O bam - - | samtools sort -@ 6 -u -O bam - | samtools markdup -@ 6 -f markdup.log - bwa.out.possorted.bam).
Fragments files were generated using Sinto (sinto fragments -b $bam -m 30 --barcodetag CB --use_chrom "(?i)^chr" --min_distance 10 --max_distance 5000 --chunksize 5000000 -p 2 -f $fragments.bed)
Per-cluster differentially accessible region were generated using pycistopic '0.1.dev214+gc72ef83.d20210522' (markers_dict = find_diff_features(cistopic_obj, imputed_acc_obj, variable='cell_type_finetuned', var_features=variable_regions, contrasts=None, adjpval_thr=0.05, log2fc_thr=np.log2(1.5), n_cpu=12)) and written to BED format using a custom python script.
Per-cluster coverage bigwigs were generated using pycistopic '0.1.dev214+gc72ef83.d20210522' (bw_paths, bed_paths = export_pseudobulk(input_data = cistopic '0.1.dev214+gc72ef83.d20210522'_obj.cell_data, variable = 'cell_type_finetuned', chromsizes = chromsizes, bed_path = bed_path, bigwig_path = bw_path, path_to_fragments = fragments_dict, n_cpu = nclusters, normalize_bigwig = True, remove_duplicates = True))
Aggregate genome coverage bigwigs were generated using DeepTools 3.5.0 (bamCoverage -b ${file} -o ${file%.bam}.RPGCnormalized.bw -bs 1 -p 4 --normalizeUsing RPGC --effectiveGenomeSize 2913022398)
For HyDrop-RNA samples (prefix HYR):
For HyDrop-ATAC samples (prefix HYA):
Barcode reads (read 2) were trimmed to exclude the intersub-barcode PCR adapters MAWK
Reads were mapped to reference genomes and expression matrices were generated using STAR version: 2.7.8a_2021-04-27 in STARsolo mode (STAR --runThreadN 6 --runMode alignReads --outSAMtype BAM SortedByCoordinate --sysShell /bin/bash --genomeDir "${star_reference_dir}" --readFilesIn "${fastq_R1_filename}" "${fastq_R2_filename}" --readFilesCommand 'gzip -c -d' --soloCBwhitelist "${whitelist_part1_filename}" "${whitelist_part2_filename}" "${whitelist_part3_filename}" --soloType CB_UMI_Complex --soloCBposition 0_0_0_9 0_20_0_29 0_40_0_49 --soloUMIposition 0_50_0_57 --sjdbGTFfile $sjdbgtf --soloCellFilter CellRanger2.2 2000 0.99 10 --soloCBmatchWLtype 1MM --outFilterMultimapNmax 1 --outSAMattributes NH HI AS nM CB UB CR CY UR UY --outFileNamePrefix ${bam_filename%bam} --outReadsUnmapped Fastx --quantMode GeneCounts --bamRemoveDuplicatesType UniqueIdentical --soloFeatures Gene GeneFull Velocyto)
For the optimisation samples (HYR__1ec4a0__20200929_Exo_S1 HYR__3ac717__20201006_non-exo_S2 HYR__70afb2__20201006_TSO-LNA_S3 HYR__c6c4b7__20201016_gtp_S4 HYR__d0a5b4__20201023_klenow_S5 HYR__f0eb5a__20201023_bst_S6), FASTQs were downsampled to match the reads per cell depth of the lowest sample and re-mapped using the same STAR parameters to generate the DOWNSAMPLED expression matrices.
Genome_build: For human samples: hg38, for mouse samples: mm10, for hybrid samples: GRCh38_GRCm10 hybrid genome, for fly samples: dm6
Supplementary_files_format_and_content: For HyDrop-ATAC samples (prefix HYA): standard tab-separated fragments files, tar archive with per-cluster differentially accessible region bed files, tar archive with per-cluster genome coverage bigwig
Supplementary_files_format_and_content: For HyDrop-RNA samples (prefix HYR): STARsolo gene expression matrix in MEX format
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| Submission date |
May 27, 2021 |
| Last update date |
Jun 04, 2021 |
| Contact name |
Stein Aerts |
| E-mail(s) |
stein.aerts@kuleuven.be
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| Organization name |
KU Leuven
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| Lab |
Lab of Computational Biology
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| Street address |
O&N4 Herestraat 49 PO Box 602
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| City |
Leuven |
| State/province |
Vlaams-Brabant |
| ZIP/Postal code |
3000 |
| Country |
Belgium |
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| Platform ID |
GPL30202 |
| Series (1) |
| GSE175684 |
HyDrop: droplet-based scATAC-seq and scRNA-seq using dissolvable hydrogel beads |
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| Relations |
| BioSample |
SAMN19370043 |
| SRA |
SRX11006913 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5343852_HYR_3ac717_20201006_non-exo_S2.DOWNSAMPLED.Solo.out.raw.tar.gz |
60.5 Mb |
(ftp)(http) |
TAR |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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