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| Status |
Public on Jun 17, 2022 |
| Title |
Single-cell RNA-seq dataset of neotenic axolotl (Batch4), NEO4, sublibrary 33 |
| Sample type |
SRA |
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| Source name |
NEO4_sublibrary_33
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| Organism |
Ambystoma mexicanum |
| Characteristics |
strain: d/d
age: Adult (1year)
tissue: brain, bladder, cloaca, eye, fore limb, hind limb, gill, gonad, pancreas, prostate, heart, intestine, kidney, liver, lung, skin, spleen, stomach, tail
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA of each tissue was purified using standard TRIzol based protocols for bulk RNA-seq;For single-cell RNA-seq, frozen cells of each tissue were thawed at 37 °C and centrifuged at 500×g for 5 min first. Then, fresh fixed or thawed frozen cells were resuspended with 100 µl PBS, and 400 µl Triton X-100 in PBS (10 µl 10% Triton X-100 in 390 µl PBS) was added. The mixture was incubated on ice for 3 min for permeabilization. Next, the cells were pelleted and resuspended with 500 µl DEPC-treated water, and 3 ml 0.1 N HCl was added slowly. After 5 min of incubation on ice, 3.5 ml Triton X-100 in Tris-HCl (35 µl 10% Triton X-100 in 3.5 ml 1 M Tris-HCl, pH 8.0) was added to quench permeabilization. Cells were then washed once with 1 ml of PBS and filtered with a 40 μm strainer. Cell density was determined using a blood cell counting chamber.
For bulk RNA-seq, RNA extraction followed reverse transcription, second strand DNA synthesis, transposase tagmentation and standard Illumina Nextera library construction. For single-cell RNA-seq, cell numbers for each tissue were estimated, and the corresponding volume of cells in PBS was split into four 96-well plates. For each well, 6.5 µl cells (from 5000 to 20000) was mixed with 0.5 µl 10 mM dNTPs (Thermo Fisher Scientific) and 0.5 µl 100 μM RT barcode primer (see Table S1). The mixture was incubated at 65 °C for 5 min and immediately placed on ice. Two microliters of 5X RT buffer (31 mM Tris-HCl, pH 8.0, 37.5 mM NaCl, 3.1 mM MgCl2, 10 mM DTT), 0.5 µl Maxima H Minus RTase (Thermo Fisher Scientific) and 0.1 µl Murine RNase inhibitor were premixed, and 2.5 µl RT mixture was added to each well. Reverse transcription was carried out by incubating the plates at gradient temperature: 3 cycles (8℃ for 12 s, 15℃ for 45 s, 20℃ for 45 s, 30℃ for 30 s, 42℃ for 2 min, and 55℃ for 3 min) and 55℃ for 60 min. After reverse transcription, plates were placed on ice for 1 min to stop the reaction. All reagents were pooled together into a 15 ml centrifuge tube and then centrifuged at 500 g for 5 min. Pellet was washed with PBS twice and resuspended with hybridization buffer (50 mM Tris-HCl, 10 mM MgCl2, 10 mM DTT, 0.1% Triton X-100, 10% PEG8000 (Sigma-Aldrich), 1% Murine RNase inhibitor). Hybridization buffer within cells was split into eight 96-well plates (3 µl for each well) and 2 µl 25 μM pre-annealing hybridization primers (50 μM HY head oligo, 50μM barcoded HY primer oligo, mixed equally and incubated at 95℃ for 2 min, then slowly cooled to 25℃ with a temperature ramp of −0.1℃/s) were added to each well (see in Table S1). Plates were incubated at 37 °C for 90 min, and 0.5 µl of 100 μM block tail primer oligo was added to block any redundant hybridization primers. After blocking for 30 min at 37 °C, all reagents were pooled into a 15 ml centrifuge tube and then centrifuged at 500 g for 5 min. The pellet was washed with PBS twice and resuspended in 40 µl of PBS. Then, 60 µl PNK mix (10 µl 10X PNK buffer (NEB), 20 µl T4 polynucleotide kinase (NEB), 10 µl 10 mM ATP (NEB), and 20 µl DEPC-treated water) was added. The PNK reaction was carried out at 37℃ for 30 min. Then, 1 ml ice-cold PBS was added to stop the reaction. Cells were then filtered using a 40μm strainer. The reagent was centrifuged at 500 g for 5 min after discarding the supernatants. The pellet was again resuspended in PBS, and the density of cells was estimated. After splitting into one or more 96-well plates. There were 5000~8000 cells per well. The volume was adjusted to 8 µl. Second strand synthesis mix (1.33 µl 10X buffer, 0.66 µl second strand synthesis enzyme mix) was added into each well and the plates were incubated at 16℃ for 3 hours (stop point at 4℃). Then, 10 µl cell lysis buffer (20 mM Tris pH 8.0, 400 mM NaCl, 100 mM EDTA, 4.4% SDS (Sangon Biotech)) and 2 µl proteinase K (Sangon Biotech) were added to the wells. Cell lysis was performed at 55℃ for 60 min. Then, 1 µl PMSF (1 mM) was added to quench the lysis reaction. The plates were incubated at 37℃ for 10 min. For each well, the mixture was then purified using 1.5X VAHTS DNA Clean Beads (Vazyme Biotech). Then, 10 µl of product in DEPC-treated water was transferred into new 96-well plates. For each plate, we randomly chose 10 wells to quantify the dsDNA concentration using Equalbit3.0. Tn5 transposase from the TruePrep DNA Library Prep Kit V2 (Vazyme Biotech) was used for cDNA tagmentation. Then, 10 µl of product in DEPC-treated water was transferred into a new 96-well plate. For each well, 14 µl PCR mix (12 µl 2X KAPA HiFi HotStart ReadyMix (Kapa Biosystems), 1 µl 10 mM P5 primer, and 1 µl 10 mM indexed P7 primer, see Table S1) was added to the plates. The PCR program was as follows: 72 °C for 5 min; 98 °C for 30 s; 12 cycles of 98 °C for 10 s, 60 °C for 30 s, and 72 °C for 1 min; 72 °C for 5 min; and a 4 °C hold. After pooling PCR products, two rounds of size selection with VAHTS DNA clean beads (Vazyme Biotech) were used to purify the DNA library between 300 and 500 bp. The libraries were finally mixed, and the concentration of dsDNA was quantified.The purified linear DNA library was circularized into single strand DNA (ssDNA) library using VAHTS® Circularization Kit for MGI (Vazyme). Then ssDNA library was amplified using DNBSEQ DNB preparation kit (MGI). Amplified DNA nanoball (DNB) was sequenced with custom primers on MGI DNBSQ-T7 with dark reaction model ( Single-cell RNA-seq: 100 cycles of read1 with dark reaction from 11-33, 100 cycles of read2;For NEO1,NEO2: PE150, no dark reaction). Human and mouse cell line libraries were sequenced with custom primers on Illumina Hiseq Xten with pair-end 150bp mode.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
DNBSEQ-T7 |
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| Description |
NA
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| Data processing |
For single cell CH-RNA-seq. Sequenced reads were trimmed for adaptor sequence.Then reads in bam files were tagged with the cell and molecular (UMI) barcode sequences using SnapTools. The reads quality under 10 were removed. Cellbarcode:In read1, the bases 1-10,19-28 (NEO3,NEO4,META1,META2); the bases 1-10,42-51(NEO1,NEO2). UMI barcode:In read1, the bases 11-18(NEO3,NEO4,META1,META2); the bases 34-41(NEO1,NEO2).The raw fastq-format sequencing data from a DNBSEQ-T7 were first split into i7 indexed sub-libraries using splitBarcode (https://github.com/MGI-tech-bioinformatics/splitBarcode). For each sub-library, the revised Drop-seq core computational tool was used for data preprocessing (http://mccarrolllab.org/wp-content/uploads/2016/03/Drop-seqAlignmentCookbookv1.2Jan2016.pdf). We used STAR (version 2.5.2a) with default parameters for mapping 3T3 cells and 293T cells. Reads from 3T3 cells and 293T cells were aligned to a merged hg19-mm10 reference genome (provided by Drop-seq group, GSE63269). To save mapping time , reads from axolotls were aligned to the axolotl genome (V3.0.0, https://www.axolotl-omics.org/assemblies) using another mapper bowtie. GTF annotation files were used to tag aligned reads. Cellular barcodes and unique molecular identifiers (UMIs) were directly extracted from Read1. A list of two rounds of cell barcode oligo sequences was used to correct the extracted cellular barcode from read1. Finally, the HTseq package was used to generate digital expression matrices of axolotl. For cell quality control of axolotl data, we excluded cells in which less than 200 transcripts were expressed. In the mixed-species experiment of 3T3 cells and 293T cells, Cellbarcode:In read1, the bases 1-10,42-51;UMI:In read1, the bases 34-41. The percentage of uniquely mapping reads for genomes of each species with over 85% UMIs assigned to one species was regarded as species-specific cells, while the remaining cells were labeled collisions. Count matrices of bulk RNA-seq of axolotl specimens were generated by featureCounts with default parameters.
For bulk RNA-seq, reads in bam file were aligned to the genome assembly using STAR version (2.5.2a) with default configurations. Next, merge the STAR alignment tagged bam were treated using DEseq2 with default configurationsand the reads are annotated with exon tags. Final featurecounts text files were used for downstream analysis.
Assembly: AmexG_v3.0.0(Axolotl genome assembly v3.0.0)(https://genome.axolotl-omics.org/cgi-bin/hgTracks?db=ambMex3&lastVirtModeType=default&lastVirtModeExtraState=&virtModeType=default&virtMode=0&nonVirtPosition=&position=AMEXG_0030000001%3A1000%2D2000&hgsid=43238_8SJNnizHOy6T42X4YaUpQyigV8RT)or(https://www.axolotl-omics.org/assemblies)
Supplementary files format and content: tab-delimited text files of digital expression matrix (dge)(single-cell RNA-seq)
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| Submission date |
Apr 25, 2022 |
| Last update date |
Jun 17, 2022 |
| Contact name |
Fang Ye |
| E-mail(s) |
11618108@zju.edu.cn
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| Phone |
+86 18167144848
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| Organization name |
Zhejiang University
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| Street address |
866 Yuhangtang Rd
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| City |
Hangzhou |
| State/province |
Zhejiang |
| ZIP/Postal code |
310058 |
| Country |
China |
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| Platform ID |
GPL30541 |
| Series (1) |
| GSE201446 |
Construction of the axolotl cell landscape using combinatorial hybridization sequencing at single cell resolution [scRNA-seq] |
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| Relations |
| BioSample |
SAMN27921170 |
| SRA |
SRX15034401 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6064459_NEO4_33_count.txt.gz |
2.3 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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