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| Status |
Public on Nov 28, 2017 |
| Title |
RT25PCR9_1002J1_p6_S3 |
| Sample type |
SRA |
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| Source name |
J1 ES cells (2i/LIF)
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| Organism |
Mus musculus |
| Characteristics |
cell type: J1 ES single-cells
strain: 129S4/SvJae
cell line: J1
Sex: male
the number of single-cell/well contained in each data: 384
method name of single-cell rna-seq: Quartz-Seq2 (v3.1, RT25)
type of sequence library preparation: Ligation
sequencer: NextSeq500
the number of fastq reads for dge-matrix (in million): 84.747886
read2 length for dge-matrix (nt): 62
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| Extracted molecule |
total RNA |
| Extraction protocol |
We performed two single-cell RNA-seq methods (Drop-seq and Quartz-Seq2). Drop-seq was performed as reported previously [Cell. 2015;161:1202–14]. Moreover, we established Quartz-Seq2 (a high-throughput single-cell RNA-sequencing method that effectively uses limited sequence reads). We used two sets of RT primer for Quartz-Seq2. The set of v3.1 RT primers has 384 kinds of unique cell barcodes, with a length of 14 nucleotides (OPC purification, FASMAC). The set of v3.2 RT primers has 1,536 kinds of unique cell barcodes, with a length of 15 nucleotides (OPC purification, Sigma). The set of v3.1 RT primers corresponds to one set of the 384-well PCR plate with lysis buffer, the wells of which have unique barcodes. The set of v3.2 RT primers corresponds to four sets of the 384-well PCR plate with lysis buffer. Single cells were isolated in the 384-well PCR plate with 1 µL of lysis buffer (0.1111 µM respective RT primers, 0.12 mM dNTP mix, 0.3% NP-40, 1 unit/µL RNasin plus) containing ERCC spike mix I.
For Quartz-Seq2, cryopreserved 384-well plates with single-cell lysate were centrifuged at 10,000 g and 4ºC for 1 min. Subsequently, we denatured total RNA in each 384 plate at 70ºC for 90 s and hybridized the RT primer to poly-adenylated RNA at 35ºC for 15 s using the C1000/S1000 thermal cycler. The resulting plates were again centrifuged at 10,000 g and 4ºC for 1 min. Next, the plates were placed on the 384 aluminum plate at 0ºC. We peeled away the seal and added 1 µL of RT premix (2x Thermopol buffer, 5 units/µL SuperScript III, 0.55 units/µL RNasin plus) to 1 µL of lysis buffer for each well using a Mantis microfluidic dispensing system (Formulatrix) or a 384 Transfer Plate system (1859-384S, Watson). The above RT solution was used for the RT25 condition. For the RT100 condition, we used the following RT solution: 2x Thermopol buffer, 20 units/µL SuperScript III, and 2.2 units/µL RNasin plus. We sealed the plates again and agitated them at 2,600 rpm and 4ºC for 1 min. The plates were then centrifuged at 10,000 g and 4ºC for 1 min. We then performed reverse transcription at 35ºC for 5 min and 50ºC for 50 min. The reverse transcription was stopped at 70ºC for 15 min. Then, the plates were placed on a prechilled aluminum block, after which we peeled off their seals. Subsequently, we turned the plates upside down on the assembled collector type A or type B (supplemental figure). We mainly used type A. We centrifuged the plates with an assemble collector at 3,010 g and 4ºC for 3 min with swing-bucket rotors. Subsequently, we collected the cDNA solution into a disposable reservoir. Typically, we obtained 650–700 µL of cDNA solution from one 384-well PCR plate. We purified and concentrated the cDNA solution using the DNA Clean & Concentrator™-5 kit (Zymo Research). We used three purification columns for one 384-well PCR plate in the case of the v3.1 RT primer system (384-cell barcode). Purified cDNA was extracted into 20 µL of nuclease-free water from one column purification and transferred into an eight-linked PCR tube (TaKaRa). The PCR tubes were placed on an aluminum PCR stand at 0ºC. We added 25 µL of TdT solution [1x Thermopol buffer, 2.4 mM dATP, 0.0384 units/µL RNase H (Invitrogen), 26.88 units/µL terminal transferase (Roche)] into 20 µL of extracted cDNA using a pipette at 0ºC. The resulting 45 µL of TdT solution was mixed with a pipette at 0ºC or ThermoMixer at 2,000 g and 0ºC for 1 min. Immediately thereafter, the PCR tubes were centrifuged at 10,000 g and 0ºC for 1 min. We used a C1000/S1000 thermal cycler equipped with the 96-Deep Well Reaction Module for the following steps. The PCR tubes were placed on the block of the thermal cycler, which had been prechilled to 0ºC. We then performed a poly-A tailing reaction at 37ºC for 75 s. The solution was inactivated at 65ºC for 10 min. The PCR tubes were placed on an aluminum PCR stand at 0ºC. We then dispensed approximately 11 µL of solution into four wells from 45 µL of TdT solution. We added 46.16 µL of PCR I premix (1.08492x MightyAmp Buffer version 2, 0.06932 µM Tagging primer, 0.05415 units/µL MightyAmp DNA polymerase) to 11 µL of TdT solution for the respective wells of the PCR tube. We performed gentle inversion mixing on the resulting solution in the PCR tube. The tubes were then centrifuged at 10,000 g and 4ºC for 1 min. Subsequently, the solution was mixed with ThermoMixer at 2,000 rpm and 4ºC for 2 min. Then, we spun down the tube again. Next, we denatured the solution at 98ºC for 130 s and hybridized Tagging primer to poly-A-tailed cDNA at 40ºC for 1 min. After that, we performed the “Increment step” by heating to 68ºC at 0.2ºC every second and performed second-strand synthesis at 68ºC for 5 min. The tubes were placed on an aluminum PCR stand at 0ºC. We added 50.232 µL of PCR II premix (0.99697x MightyAmp Buffer version.2, 1.8952 µM gM primer) to 56.16 µL of PCR I solution. We performed gentle inversion mixing on the resulting solution in the PCR tube. The tubes were then centrifuged at 10,000 g and 4ºC for 1 min. Subsequently, the solution was mixed with ThermoMixer at 2,000 rpm and 4ºC for 2 min, after which we spun down the tube again. We then placed it on the block of the thermal cycler at 68ºC. Subsequently, we amplified the cDNA for 11 cycles under the following conditions: 98ºC for 10 s, 65ºC for 15 s, and 68ºC 5 min. We then incubated the tube at 68ºC for an additional 5 min. Finally, we transferred all of the PCR solution, derived from one 384-well PCR plate, to a 50-mL Polypropylene Centrifuge Tube (Watson). Typically, we obtained approximately 1.2 mL of PCR solution per 384-well PCR plate. We added 32 µL of 3 M sodium acetate (pH 5.2) and 6420 µL PB-Buffer (Qiagen) to the PCR solution. The mixture was then purified using a MinElute Spin Column (Qiagen). Purified cDNA was extracted into 40 µL of nuclease-free water. We additionally purified the cDNA with 32 µL of Ampure XP beads. Finally, we obtained 32 µL of purified cDNA. We checked the length distribution of amplified cDNA with an Agilent High Sensitivity DNA Kit (Agilent). The typical average size of the amplified cDNA in Quartz-Seq2 was approximately 1,400 bp. In the case of the usage of the v3.2 RT primer in Quartz-Seq2, we modified the above steps as follows. After reverse transcription, we collected cDNA solution into a disposable reservoir from four sets of 384-well plates, which corresponded to 1,536 wells. We purified and concentrated the cDNA solution using eight purification columns for four 384-well PCR plates in the case of the v3.2 RT primer system. In the PCR step, we amplified cDNA for nine cycles with the following conditions: 98ºC for 10 s, 65ºC for 15 s, and 68ºC for 5 min. Finally, we transferred all of the PCR solution derived from four 384-well plates to a 50-mL Polypropylene Centrifuge Tube (Watson). Typically, we obtained approximately 3.5 mL of PCR solution per four 384-well PCR plates. We added 88 µL of 3 M sodium acetate (pH 5.2) and 17.6 mL of PB-Buffer (Qiagen) to the PCR solution. The mixture was purified using the MinElute Spin Column (Qiagen). Subsequently, cDNA was again purified using Ampure XP magnetic beads. For the Quartz-Seq1-like reaction, we performed the following procedure in accordance with our previous study. We added 1 µL of RT premix (2x PCR buffer, 5 units/µL SuperScript III, 0.55 units/µL RNasin plus) to 1 µL of lysis buffer. We then performed reverse transcription at 35ºC for 5 min and 45ºC for 20 min. This reverse transcription was stopped at 70ºC for 15 min. We added 5 µL of ExoIB solution (1.6x Exonuclease I buffer, 3.2x PCR buffer, 16 mM DTT) and 20 µL of TdT solution [1x PCR buffer, 3 mM dATP, 0.0384 units/µL RNase H (Invitrogen), 33.6 units/µL terminal transferase (Roche)] into 20 µL of extracted cDNA using a pipette at 0ºC. The PCR tubes were placed on the block of the thermal cycle
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
We analyzed single-cells, which were sorted to 384-wells in total.
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| Data processing |
BCL files generated by Illumina NextSeq500 were converted to fastq files by bcl2fastq2 (v2.17.1.14) with demultiplexing pool barcodes.
Data for Quartz-Seq2 was processed by Quartz-Seq2 pipeline. Data for Drop-seq was processed by Drop-seq pipeline.
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include UMI counts for each gene for each cell
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| Submission date |
Nov 28, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Itoshi NIKAIDO |
| E-mail(s) |
itoshi.nikaido@riken.jp
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| Organization name |
RIKEN
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| Department |
Center for Biosystems Dynamics Research
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| Lab |
Laboratory for Bioinformatics Research
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| Street address |
2-1 Hirosawa
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| City |
Wako |
| State/province |
Saitama |
| ZIP/Postal code |
351-0198 |
| Country |
Japan |
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| Platform ID |
GPL19057 |
| Series (1) |
| GSE99866 |
Quartz-Seq2: a high-throughput single-cell RNA-sequencing method that effectively uses limited sequence reads |
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| Relations |
| BioSample |
SAMN08104813 |
| SRA |
SRX3426323 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2866805_RT25PCR9_1002J1_p6_S3_Mmusculus_Mmusculus_seqlev_d2_dge.txt.gz |
2.8 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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