GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
Sample GSM4454017 Query DataSets for GSM4454017
Status Public on Nov 10, 2021
Title SPM
Sample type SRA
Source name HEK 293T/ murine brown preadipocyte line
Organisms Homo sapiens; Mus musculus
Characteristics culture method: In vitro cell culture
passages: 25-30
cell line: mixture of HEK 293T and iBA
Growth protocol The human embryonic kidey 293T and murine brown preadipocyte line (iBA) cell lines were cultured to 90% confluency in Glutamax DMEM supplemented with FBS and penicillin-streptomycin.
Extracted molecule total RNA
Extraction protocol 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). 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
Data processing 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).
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: mm10-hg19 mixed genome GSE63269
Supplementary_files_format_and_content: tab-delimited text files include read-count matrices and digital gene expression files for each species
Submission date Apr 05, 2020
Last update date Nov 10, 2021
Contact name Marjan Biočanin
Organization name EPFL
Department SV-IBI
Lab Laboratory for systems biology and genetics/UPDE
Street address Station 19, SV 3820
City Lausanne
State/province Vaud
ZIP/Postal code 1015
Country Switzerland
Platform ID GPL19415
Series (2)
GSE148092 Low-input, deterministic profiling of single-cell transcriptomes reveals individual intestinal organoid subtypes comprised of single, dominant cell types [SPM]
GSE148093 Low-input, deterministic profiling of single-cell transcriptomes reveals individual intestinal organoid subtypes comprised of single, dominant cell types
BioSample SAMN14540514
SRA SRX8060123

Supplementary file Size Download File type/resource
GSM4454017_SPM.tar.gz 527.8 Kb (ftp)(http) TAR
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap