GEO help: Mouse over screen elements for information.
|Public on Nov 10, 2021
|culture method: In vitro cell culture
cell line: HEK 293T
|The human embryonic kindley 293T cell line was cultured to 90% confluency in Glutamax DMEM supplemented with FBS and penicillin-streptomycin.
|Prior to use, cells were washed with PBS, dissociated via Trypsin-EDTA, washed with PBS, BSA and counted with Trypan blue live-dead stain using Countess cell counter (Invitrogen). Cells were mixed in a 1:1 ratio, adjusted to 20 cells/µl, and re-suspended in a cell buffer containing PBS, BSA, and Optiprep previous to loading onto the DisCo chip.
After bead-cell in droplet co-encapsulation, the gel loading tip containing the sample droplets was transferred to a bead collection chip inlet (cp-chip; as described in Lab on a chip). Droplets in the tip were flushed to bead collection chip. Subsequently to bead capture, washing was performed as in Drop-seq with SSC and Reverse transcription buffer directly in the recovery chip. Reverse transcription solution was added to the beads and the recovery chip was placed on a heating block to perform first strand cDNA synthesis (RT) for 90 minutes at 42oC. After RT reaction beads were washed on the recovery chip with TE-SDS once, with TE-TW twice and with Tris once. The beads were treated with Exonuclease I for 45 minutes at 37oC to remove single-stranded oligonucleotides on beads. After Exonuclease I treatment, beads were washed with TE-SDS once, with TE-TW twice (as after reverse transcription). Beads were then eluted from the recovery chip in dH2O. cDNA was amplified for 18 – 23 cycles using Kapa HiFi Hot start ready mix. cDNA was purified with CleanPCR magnetic beads using 0.6X ratio to remove small cDNA fragments and primers. To assess the cDNA yield and quality concentration was measured using Qubit and cDNA traces quality was assessed using Fragment Analyzer (Agilent). cDNA was tagmented with in-house Tn5 (generated as described in Picelli et al..) and the library was purified using CleanPCR magnetic beads (0.6X ratio).
Libraries were sequenced on a NextSeq 500 system (Illumina) following recommendations from original protocol (20 bp for read 1 and 50 bp for read2).
|Illumina NextSeq 500
|Drop-seq tools 2.3.0 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)
Supplementary_files_format_and_content: tab-delimited text files include read-count matrices for each experiment and digital gene expression files
|Apr 05, 2020
|Last update date
|Nov 10, 2021
|Laboratory for systems biology and genetics/UPDE
|Station 19, SV 3820
|Low-input, deterministic profiling of single-cell transcriptomes reveals individual intestinal organoid subtypes comprised of single, dominant cell types [Dispencell]
|Low-input, deterministic profiling of single-cell transcriptomes reveals individual intestinal organoid subtypes comprised of single, dominant cell types