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| Status |
Public on Nov 10, 2021 |
| Title |
Time5_HEK_180mins |
| Sample type |
SRA |
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| Source name |
HEK 293T
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK 293T
sample type: In vitro cell culture
passage: 25-30 passage
condition: 180min_on_ice
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| Growth protocol |
The human embryonic kidney 293T cell line was cultured in suspension in a DMEM medium supplemented 10% fetal bovine serum and 1% antibiotics (L-Glutamine-penicillin-streptomycin).
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Recovered cells were centrifuged at 600g for 5 min and supernatant was replaced with 25 µl of PBS containing 0.01 % bovine serum albumin (Sigma). Subsequently 25 ml of lysis buffer were added, containing 0.1 % Sarkosyl (Sigma), 5 mM EDTA (Life Technologies), 0.1 M Tris (pH 7.5, Sigma) and 25 mM 1,4-Dithioreitol (DTT, Sigma), 800 units/ml RNase inhibitor (NEB) and 500 Drop-Seq beads (Beads, Lot 120817, ChemGenes). All beads pelleting steps were carried out at 1000 g for 1 min in 1.5 ml microtubes (Axygen). Reverse transcription (RT), exonuclease I (ExoI) treatment and PCR were performed as described Macosko et al. with minor adaptations30. Lysed cells were incubated at 1400 rpm for 5 min at room temperature and subsequently washed twice with 1 ml of 6x SSC buffer (Sigma).
Reverse transcription was performed for 90 min at 42°C in 50 µl of 1 mM dNTPs (Clontech), 2.5 µM template switch oligo (see Table 1), 1250 units/ml RNase inhibitor, 1x Maxima RT buffer and 10,000 units/ml Maxima H minus reverse transcriptase (Thermo Fisher Scientific). Drop-seq beads were washed twice with 0.5 % SDS (Applichem) in 10 mM Tris (TE‑SDS), twice with 0.01 % Tween‑20 (Sigma) in 10 mM Tris (TE‑TW) and once with 10 mM Tris pH 7.5. The Drop-seq beads pellet was then incubated with 50 µl of exonuclease mix containing 1x Exonuclease I Buffer and 1000 units/ml Exonuclease I (NEB), and incubated at 37°C for 45 min at 1,400 rpm). Drop-seq beads were washed twice with TE‑SDS, twice with TE‑TW and once with double-distilled H2O. Beads were amplified by PCR in 25 µL of 1x Hifi HotStart Readymix (Kapa Biosystems) and 0.8 µM TSO-PCR primer (Table 1) at 95 C 3 min; 4 cycles of: 98 C for 20 sec, 65 C for 45 sec, 72 C for 3 min; then 16 cycles of: 98°C for 20 sec, 67°C for 20 sec, 72°C for 3 min and an extension step of 5 min. Libraries were purified using Ampure XP beads (0.6x ratio to remove small fragments), cDNA was quantified using a Qubit HS kit (Thermo Fisher Scientific) and integrity analyzed on a Fragment Analyzer (Agilent). Libraries were prepared using in-house produced Tn5 loaded with adapters, as described31. Size selected and purified libraries were sequenced paired-end on a NextSeq 500 system (Illumina) in High-Output mode following recommendations from the original protocol (read 1 20 bp and read 2 50 bp)
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Drop-seq tools package on the EPFL SCITAS HPC platform. After trimming and sequence tagging, reads were aligned to the mouse reference genome (mm10) using STAR (version 2.7.0.e). https://github.com/mccrowjp/Dropseq
Following the alignment, the gene annotation was added, bead synthesis errors were corrected and cell barcodes extracted. Subsequently, the BAM files containing the processed data were used to obtain digital gene expression matrices.
Downstream data analysis was carried out using R (version 3.5.0)
Genome_build: hs.g38r91
Supplementary_files_format_and_content: tab-delimited text files include read-count matrices for each experiment and digital gene expression files
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| Submission date |
Sep 08, 2021 |
| Last update date |
Nov 10, 2021 |
| Contact name |
Marjan Biočanin |
| E-mail(s) |
marjan.biocanin@epfl.ch
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| Organization name |
EPFL
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| Department |
SV-IBI
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| Lab |
Laboratory for systems biology and genetics/UPDE
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| Street address |
Station 19, SV 3820
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| City |
Lausanne |
| State/province |
Vaud |
| ZIP/Postal code |
1015 |
| Country |
Switzerland |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE148093 |
Low-input, deterministic profiling of single-cell transcriptomes reveals individual intestinal organoid subtypes comprised of single, dominant cell types |
| GSE183689 |
Low-input, deterministic profiling of single-cell transcriptomes reveals individual intestinal organoid subtypes comprised of single, dominant cell types [HEK Kinetic] |
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| Relations |
| BioSample |
SAMN21357881 |
| SRA |
SRX12105433 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5567779_Time5_HEK_180mins_S5.txt.gz |
259.2 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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