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| Status |
Public on Jun 04, 2021 |
| Title |
HYA__combined__20210323_cortex_phu_dv_etssb_1-5_S5 |
| Sample type |
SRA |
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| Source name |
Mouse cortex nuclei
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
tissue/cell line: Cerebral cortex
developmental stage: Mouse: P56
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| Growth protocol |
Female wild-type P56 BL/6 mice were kept according to standard animal husbandry practices were maintained in a specific pathogen-free facility under standard housing conditions with continuous access to food and water. Mice used in the study were 1–8-weeks old and were maintained on 14 h light, 10 h dark light cycle from 7 to 21 h.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cortex was separated from female P56 BL/6 mouse brains, immediately snap-frozen in liquid nitrogen and preserved in -80 °C. A ~1 cm3 frozen piece of mouse cortex tissue was transferred to 1 mL of ice-cold homogenisation buffer (320 mM Sucrose, 10 mM NaCl, 10 mM Tris-HCl pH 7.5, 3 mM MgAcetate, 1 mM DTT, 0.1 mM EDTA, 0.1% IGEPAL, 1X cOmplete Protease Inhibitor Cocktail) in a Dounce homogenizer mortar and thawed for 3 minutes. The tissue was homogenised with 10 strokes of pestle A and 5 strokes of pestle B until a homogeneous cell suspension was achieved. The resulting homogenate was filtered through a 40 μm Flowmi strainer. The mortar was washed with 1.65 mL of ice-cold homogenisation buffer to collect any remaining cells. 2.65 mL of ice-cold gradient medium was added to 1.65 mL of homogenate. 4 mL of 29% iodoxanol cushion (129.2 mM Sucrose, 77.5 mM KCl, 15.5 mM MgCl, 31 mM Tris-HCl pH 7.5, 29% Iodoxanol) was loaded into an ultracentrifuge. 5.3 mL of sample was gently layered on top of the 29% iodoxanol cushion without mixing both phases. Sample was centrifuged at 10 000 xg, 4°C for 30 minutes and the supernatant was gently removed without disturbing the nuclei pellet. Nuclei were resuspended in 100 μL of Nuclei buffer. 50 000 nuclei were resuspended in 50 μL of ATAC Reaction Mix (10% DMF, 10% Tris-HCl pH 7.4, 5 mM MgCl2, 5 ng/uL Tn5, 70 uM Pitstop, 0.1% Tween-20, 0.01% Digitonin) and incubated at 37 °C for 1 hour without shaking. 100 μL of 0.1% BSA in PBS was added and the nuclei were pelleted at 500 xg, 4 °C for 5 minutes and resuspended in 40 μL of 0.1% BSA in PBS. Tagmented nuclei were added to 100 μL of PCR mix (1.3X Phusion HF Buffer, 15% Optiprep, 1.3 mM dNTPs, 39 mM DTT, 0.065 U/uL Phusion HF Polymerase, 0.065 U/uL Deep Vent Polymerase, 0.013 U/uL ET SSB). PCR mix was co-encapsulated with 35 μL of freshly thawed HyDrop-ATAC beads in RAN oil on the Onyx microfluidics platform. The resulting emulsion was collected in aliquots of 50 μL total volume and thermocycled according to the PCR1 program (72 °C 15 min., 98 °C 3 min., 13 PCR cycles of [98 °C 10 s, 63 °C 30 s, 72 °C 1 min.], followed by a final hold on 4 °C). 125 μL of recovery agent (20% PFO in HFE), 55 μL of GITC Buffer (5 M GITC, 25 mM EDTA, 50 mM Tris-HCl pH 7.4) and 5 μL of 1 M DTT was added to each separate aliquot of 50 μL thermocycled emulsion and incubated on ice for 5 minutes. 5 μL of Dynabeads was added to the aqueous phase and incubated for 10 minutes. Dynabeads were pelleted on a Nd magnet and washed twice with 80% EtOH. Elution was performed in 50 μL of EB-DTT-Tween (10 mM DTT, 0.1% Tween-20 in EB). A 1X Ampure bead purification was performed according to manufacturer’s recommendations. Elution was performed in 30 μL of EB-DTT (10 mM DTT in EB).
Eluted library was further amplified in 100 μL of PCR2 mix (1X KAPA HiFi, 1 uM index i7 primer, 1 uM index i5 primer). Final library was purified in a 0.4X-1.2X double-sided Ampure purification and eluted in 25 μL of EB-DTT (10 mM DTT in EB).
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
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| Description |
processed data file: HYA__combined__20210323_cortex_phu_dv_etssb.cluster_bigwigs.tar.gz
processed data file: HYA__combined__20210323_cortex_phu_dv_etssb.cluster_dars.tar.gz
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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 |
GPL30172 |
| Series (1) |
| GSE175684 |
HyDrop: droplet-based scATAC-seq and scRNA-seq using dissolvable hydrogel beads |
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| Relations |
| BioSample |
SAMN19370081 |
| SRA |
SRX11006910 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5343849_HYA_combined_20210323_cortex_phu_dv_etssb_1-5_S5.sinto.fragments.tsv.gz |
449.9 Mb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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