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Sample GSM3770929 Query DataSets for GSM3770929
Status Public on Apr 12, 2020
Title HEK293T, NIH/3T3, sci-fate
Sample type SRA
 
Source name cell line
Organisms Homo sapiens; Mus musculus
Characteristics cell line: HEK293T, NIH/3T3
treatment: 100 nM DEX for 0h, 2h, 4h, 6h, 8h and 10h before harvest
Treatment protocol A549 cells were treated with 100 nM DEX for 0h, 2h, 4h, 6h, 8h and 10h before harvest. Control cells were treated with same volume of EtOH. Cells in all treatment conditions were incubated with 200uM S4U for the last two hours before cell harvest. For HEK293T and NIH/3T3 cells, cells were incubated with 200uM S4U for 6 hours before cell harvest.
Growth protocol All mammalian cells were cultured at 37°C with 5% CO2, and were maintained in high glucose DMEM (Gibco cat. no. 11965) for HEK293T and NIH/3T3 cells or DMEM/F12 medium for A549 cells, both supplemented with 10% FBS and 1X Pen/Strep (Gibco cat. no. 15140122; 100U/ml penicillin, 100µg/ml streptomycin). Cells were trypsinized with 0.25% typsin-EDTA (Gibco cat. no. 25200-056) and split 1:10 three times a week.
Extracted molecule polyA RNA
Extraction protocol All cell lines (A549, HEK293T and NIH/3T3 cells) were trypsinized, spun down at 300xg for 5 min (4°C) and washed once in 1X ice-cold PBS. All cells were fixed with 4ml ice cold 4% paraformaldehyde (EMS) for 15 min on ice. After fixation, cells were pelleted at 500xg for 3 min (4°C) and washed once with 1ml PBSR (1 x PBS, pH 7.4, 1% BSA, 1% SuperRnaseIn, 1% 10mM DTT). After wash, cells were resuspended in PBSR at 10 million cells per ml, and flash frozen and stored in liquid nitrogen.
Paraformaldehyde fixed cells were thawed on 37°C water bath, spun down at 500xg for 5 min, and incubated with 500ul PBSR including 0.2% Triton X-100 for 3min on ice. Cells were pelleted and resuspended in 500ul nuclease free water including 1% SuperRnaseIn. 3ml 0.1N HCl were added into the cells for 5min incubation on ice. 3.5ml Tris-HCl (pH = 8.0) and 35ul 10% Triton X-100 were added into cells to neutralize HCl. Cells were pelleted and washed with 1ml PBSR. Cells were resuspended in 100ul PBSR. 100ul PBSR with fixed cells were incubated with mixture including 40ul Iodoacetamide (IAA, 100mM), 40ul sodium phosphate buffer (500mM, pH = 8.0), 200ul DMSO and 20ul H2O, at 50°C for 15min. The reaction was quenched by 8ul DTT (1M) and 8.5ml PBS. Cells were pelleted and resuspended in 100ul PBSI (1 x PBS, pH 7.4, 1% BSA, 1% SuperRnaseIn). For all later washes, nuclei were pelleted by centrifugation at 500xg for 5 min (4°C). The following steps are similar with sci-RNA-seq protocol with paraformaldehyde fixed nuclei. Briefly, cells were distributed into four 96-well plates. For each well, 5,000 nuclei (2 µL) were mixed with 1 µl of 25 µM anchored oligo-dT primer (5′-ACGACGCTCTTCCGATCTNNNNNNNN[10bp index]TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3′, where “N” is any base and “V” is either “A”, “C” or “G”; IDT) and 0.25 µL 10 mM dNTP mix (Thermo), denatured at 55°C for 5 min and immediately placed on ice. 1.75 µL of first-strand reaction mix, containing 1 µL 5X Superscript IV First-Strand Buffer (Invitrogen), 0.25 µl 100 mM DTT (Invitrogen), 0.25 µl SuperScript IV reverse transcriptase (200 U/μl, Invitrogen), 0.25 μL RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen), was then added to each well. Reverse transcription was carried out by incubating plates at the following temperature gradient: 4°C 2 minutes, 10°C 2 minutes, 20°C 2 minutes, 30°C 2 minutes, 40°C 2 minutes, 50°C 2 minutes and 55°C 10 minutes. All cells (or nuclei) were then pooled, stained with 4',6-diamidino-2-phenylindole (DAPI, Invitrogen) at a final concentration of 3 μM, and sorted at 25 nuclei per well into 5 μL EB buffer. Cells were gated based on DAPI stain such that singlets were discriminated from doublets and sorted into each well. 0.66 μl mRNA Second Strand Synthesis buffer (NEB) and 0.34 μl mRNA Second Strand Synthesis enzyme (NEB) were then added to each well, and second strand synthesis was carried out at 16°C for 180 min. Each well was then mixed with 5 μL Nextera TD buffer (Illumina) and 1 μL i7 only TDE1 enzyme (25 nM, Illumina, diluted in Nextera TD buffer), and then incubated at 55°C for 5 min to carry out tagmentation. The reaction was stopped by adding 12 μL DNA binding buffer (Zymo) and incubating at room temperature for 5 min. Each well was then purified using 36 uL AMPure XP beads (Beckman Coulter), eluted in 16 μL of buffer EB (Qiagen), then transferred to a fresh multi-well plate. For PCR reactions, each well was mixed with 2μL of 10 μM P5 primer (5′-AATGATACGGCGACCACCGAGATCTACAC[i5]ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′; IDT), 2 μL of 10 μM P7 primer (5′-CAAGCAGAAGACGGCATACGAGAT[i7]GTCTCGTGGGCTCGG-3′; IDT), and 20 μL NEBNext High-Fidelity 2X PCR Master Mix (NEB). Amplification was carried out using the following program: 72°C for 5 min, 98°C for 30 sec, 18-22 cycles of (98°C for 10 sec, 66°C for 30 sec, 72°C for 1 min) and a final 72°C for 5 min. After PCR, samples were pooled and purified using 0.8 volumes of AMPure XP beads. Library concentrations were determined by Qubit (Invitrogen) and the libraries were visualized by electrophoresis on a 6% TBE-PAGE gel. Libraries were sequenced on the NextSeq 500 platform (Illumina) using a V2 150 cycle kit (Read 1: 18 cycles, Read 2: 130 cycles, Index 1: 10 cycles, Index 2: 10 cycles).
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
 
Description sci-fate library
Data processing For sc-RNA-seq processing, read alignment and gene count matrix generation for single cell RNA-seq was the same with sci-RNA-seq ("J. Cao et al., Comprehensive single cell transcriptional profiling of a multicellular organism by combinatorial indexing. bioRxiv, 104844 (2017).") with minor modifications: Reads were mapped to a chimeric reference genome of human (hg19) and mouse (mm10) for RNA-seq from A594, HEK293T and NIH/3T3 cells, and mouse (mm10) only for RNA-seq from mouse kidney experiment with STAR/v 2.5.2b, with gene annotations (GENCODE V19 for human; GENCODE VM11 for mouse). The transcriptome barcode information for each cell or treatment conditions are listed in the cell annotation file of processed data. For mixed-species experiment, the percentage of uniquely mapping reads for genomes of each species was calculated. Cells with over 80% of UMIs assigned to one species were regarded as species-specific cells, with the remaining cells classified as mixed cells or “collisions”.
The single cell sam files were first converted into alignment tsv file using sam2tsv function in jvarkit. Next, for each single cell alignment file, mutations matching the background SNPs were filtered out. For background SNP reference of A549 cells, we downloaded the paired-end bulk RNA-seq data for A549 cells from ENCODE (sampled name: ENCFF542FVG, ENCFF538ZTA, ENCFF214JEZ, ENCFF629LOL, ENCFF149CJD, ENCFF006WNO, ENCFF828WTU, ENCFF380VGD). Each paired-end fastq files were first adaptor-clipped using trim_galore/0.4.1 with default settings, aligned to human hg19 genome build with STAR/v2.5.2b. Unmapped and multiple mapped reads were removed by samtools/v1.3. Duplicated reads were filtered out by MarkDuplicates function in picard/1.105. De-duplicated reads from all samples were combined and sorted with samtools/v1.3. Background SNPs were called by mpileup function in samtools/v1.3 and mpileup2snp function in VarScan/2.3.9. For HEK293T and NIH/3T3 test experiment, background SNP reference was generated in a similar pipeline above, with the aggregated single cell sam data from control condition (no S4U labeling and no IAA treatment condition). For each single cell alignment file, all mutations with quality score <= 13 were removed. Mutations at the both ends of each reads were mostly due to sequencing errors, and thus also were filtered out. For each read, we checked if there are T > C mutations for sense strand or A > G mutations for antisense strand, and labeled these mutated reads as newly synthesized. Each cell was characterized by two digital gene expression matrixes from the full sequencing data and newly synthesized RNA data as described above.
Genome_build: human reference genome (hg19), mouse reference genome (mm10)
Supplementary_files_format_and_content: Processed data files are in txt format including a cell annotation file, gene annotation file, and a gene count sparse matrix for boh full sequencing data and newly synthesized RNA data
HEK293T_3T3_cell_annotate.txt: Cell annotation file for full transcriptome data: sample is cell id of each single cell with the reverse transcription barcode attached; exon_reads are number of UMIs mapping to exon region, intron_reads are number of UMIs mapping to intron region, all_reads are number of total UMIs, human_reads are number of UMIs mapping to human genome, mouse_reads are number of UMIs mapping to mouse genome, source are the human/mouse source of cells based on the ratio of human UMIs
HEK293T_3T3_gene_annotate.txt: Gene annotation file for full transcriptome data including gene id, gene typeand gene short name.
HEK293T_3T3_gene_count.txt: A sparse matrix file for full transcriptome data: each row corresponds to gene id (with gene information in the HEK293T_3T3_gene_annotate.txt; each column corresponds to each cell (with cell source information in the HEK293T_3T3_cell_annotate.txt); each value in the matrix corresponds to UMI count.
HEK293T_3T3_cell_annotate_newly_synthesised.txt: Cell annotation file for newly synthesized transcriptome data: sample is cell id of each single cell with the reverse transcription barcode attached; exon_reads are number of UMIs mapping to exon region, intron_reads are number of UMIs mapping to intron region, all_reads are number of total UMIs.
HEK293T_3T3_gene_annotate_newly_synthesised.txt: Gene annotation file for newly synthesized transcriptome data including gene id, gene typeand gene short name.
HEK293T_3T3_gene_count_newly_synthesised.txt: A sparse matrix file for newly synthesized transcriptome data: each row corresponds to gene id (with gene information in the HEK293T_3T3_gene_annotate_newly_synthesised.txt; each column corresponds to each cell (with cell source information in the HEK293T_3T3_cell_annotate_newly_synthesised.txt); each value in the matrix corresponds to UMI count.
A549_cell_annotate.txt: Cell annotation file for full transcriptome data: sample is cell id of each single cell with the reverse transcription barcode attached; all_exon are number of UMIs mapping to exon region, all_intron are number of UMIs mapping to intron region, all_reads are number of total UMIs, treatment_time is the DEX treatment time, and doublet_score is the doublet score calculated per cell from the doublet analysis.
A549_gene_annotate.txt: Gene annotation file for full transcriptome data including gene id, gene typeand gene short name.
A549_gene_count.txt: A sparse matrix file for full transcriptome data: each row corresponds to gene id (with gene information in the A549_gene_annotate.txt; each column corresponds to each cell (with cell source information in the A549_cell_annotate.txt); each value in the matrix corresponds to UMI count.
A549_cell_annotate_newly_synthesised.txt: Cell annotation file for newly synthesized transcriptome data: sample is cell id of each single cell with the reverse transcription barcode attached; all_exon are number of UMIs mapping to exon region, all_intron are number of UMIs mapping to intron region, all_reads are number of total UMIs, treatment_time is the DEX treatment time, and doublet_score is the doublet score calculated per cell from the doublet analysis.
A549_gene_annotate_newly_synthesised.txt: Gene annotation file for newly synthesized transcriptome data including gene id, gene typeand gene short name.
A549_gene_count_newly_synthesised.txt: A sparse matrix file for newly synthesized transcriptome data: each row corresponds to gene id (with gene information in the A549_gene_annotate_newly_synthesised.txt; each column corresponds to each cell (with cell source information in the A549_cell_annotate_newly_synthesised.txt); each value in the matrix corresponds to UMI count.
 
Submission date May 16, 2019
Last update date Apr 13, 2020
Contact name Junyue Cao
E-mail(s) cao1025@uw.edu
Organization name University of Washington
Department Department of Genome Sciences
Lab Shendure lab
Street address Foege Building S-210, 3720 15th Ave NE
City Seattle
State/province WA
ZIP/Postal code 98195-5065
Country USA
 
Platform ID GPL19415
Series (1)
GSE131351 Characterizing the temporal dynamics of gene expression in single cells with sci-fate
Relations
BioSample SAMN11661078
SRA SRX5848247

Supplementary file Size Download File type/resource
GSM3770929_HEK293T_3T3_cell_annotate.txt.gz 176.8 Kb (ftp)(http) TXT
GSM3770929_HEK293T_3T3_cell_annotate_newly_synthesised.txt.gz 56.5 Kb (ftp)(http) TXT
GSM3770929_HEK293T_3T3_gene_annotate.txt.gz 1006.9 Kb (ftp)(http) TXT
GSM3770929_HEK293T_3T3_gene_annotate_newly_synthesised.txt.gz 1006.9 Kb (ftp)(http) TXT
GSM3770929_HEK293T_3T3_gene_count.txt.gz 68.2 Mb (ftp)(http) TXT
GSM3770929_HEK293T_3T3_gene_count_newly_synthesised.txt.gz 10.9 Mb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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