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Sample GSM7825047 Query DataSets for GSM7825047
Status Public on Oct 10, 2023
Title huam_mouse_mix
Sample type SRA
Source name 293T and 3T3
Organisms Homo sapiens; Mus musculus
Characteristics cell line: 293T and 3T3
cell type: human embryonic kidney cell line and mouse embryonic fibroblasts
genotype: WT
treatment: encapsulated in hydrogels
Treatment protocol We dissolved 0.0458 g of Dextran (Yeasen, MW 500K, 61216ES25) and 0.0353 g of PEGDA (Sigma-Aldrich, MW 6K, 701963) in 700 μL of 1 × DPBS. To this solution, we added 100 μL of 4 % (w/v) LAP (Sigma-Aldrich, 900889) and 30 μL of PEGDA (Sigma-Aldrich, MW 575, 437441), finally adjusting the volume to 1 mL with 1 × DPBS. This mixture underwent centrifugation at 16,000g for 30 minutes, inducing liquid-liquid phase separation. Following centrifugation, a clear separation boundary was observed between the upper PEGDA-rich phase and the lower dextran-rich phase. For cell manipulation, we resuspended 1.79 million mammalian cells in 200 μL of the dextran-rich phase using a microfluidic device with a 40 μm height. In our microfluidic system, we used a 3 mL BD syringe for a 2% FS10 (w/w) solution in HFE-7500 (Tunderbio, FSA2). Additionally, two 1 mL BD syringes were allocated for the PEGDA-rich phase and the dextran-rich phase containing cells. These preloaded syringes were securely positioned within the injection pump (Leadfluid, TFD04). Flow rates in the microfluidic system were set as follows: 6.67 μL/min for the oil phase, 1.11 μL/min for the PEGDA-rich phase, and 1.55 μL/min for the Dextran-rich phase. Approximately 200 μL of collected droplets were then subjected to 2 minutes of UV irradiation and subsequently treated with 20%(v/v) PFO in HFE-7500 (Sigma-Aldrich, 370533-25G) to form HetHydrogels.
Growth protocol Human 293T (ATCC) and Mouse 3T3 cells (ATCC) were maintained in DMEM media supplemented with 10 % fetal bovine serum in humidified at mosphere with 5 % CO2 at 37 ℃. Cells were harvested from a culture dish, underwent a single wash with 1×PBS, and were subsequently quantified using using the Trypan Blue method[37]. Before encapsulation, cells were suspended in a 4.58 % (w/v) dextran solution at a concentration of 1.79 million cells per 200 μL when utilizing a microfluidic device with a 40 μm height. All centrifugation steps were executed at 300 × g for 3 minutes at 4 °C.
Extracted molecule genomic DNA
Extraction protocol Lysis Buffer 1 involved immersing 50 µL of the HetHydrogels in 200 µL of TCL lysis buffer (QIAGEN,1031576) supplemented with 1% (v/v) beta-mercaptoethanol (Millipore,444203), followed by a 5-minute room temperature incubation. Lysis Buffer 2 comprised the placement of 50 µL of the HetHydrogels into 200 µL of lysis buffer containing 50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 20 µg/mL Proteinase K (Thermo Scientific, EO0491), and 0.2% SDS, with a subsequent 30-minute incubation at 55°C. Lysis Buffer 3 involved submerging 50 µL of the HetHydrogels in 200 µL of lysis buffer containing 0.1% Triton X-100, 10 mM Tris-HCl (pH 8.0), and 20 µg/mL Proteinase K (Thermo Scientific, EO0491), followed by a 30-minute incubation at 55°C. Lastly, Lysis Buffer 4 consisted of 50 µL of the HetHydrogels in 200 µL of lysis buffer composed of 0.1% Triton X-100, 10 mM Tris-HCl (pH 8.0), and 20 µg/mL Proteinase K (Thermo Scientific, EO0491), with a 30-minute incubation at 55 °C. Additionally, for Lysis Buffer 4, the composition included 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% (vol/vol) NP-40, and 1% (vol/vol) BSA, with an incubation step on ice for 3 minutes.
This step is carried out following the standard procedures outlined in the DNBelab C-Series High-Throughput Single-Cell ATAC Library Preparation Kit instruction manual (BGI, 940-000793-00).
Library strategy ATAC-seq
Library source genomic single cell
Library selection other
Instrument model Illumina NovaSeq 6000
Data processing raw paired-end reads were aligned to the hg38 or hg38_mm10 composite reference genome using Chromap
alignment files were subjected to barcode merging using D2C
MergeBamAlignment was employed to consolidate the data, followed by sorting using the "samtools sort" command.
mtDNA coverage was assessed using "samtools depth."
Peak calling was conducted using the MACK2.
Assembly: hg38 or hg38_mm10
Supplementary files format and content: hg38.bam and depth.txt for diffferent lysis conditions
Supplementary files format and content: 293T_percellaverage_sorted.bam and 3T3_percellaverage_sorted.bam for scATAC-seq data within Hydrogles
Submission date Oct 05, 2023
Last update date Oct 10, 2023
Contact name Guoqiang Zhou
Organization name Fudan University School of Life Sciences
Street address Fudan University, Shanghai, 200438, China
City shanghai
ZIP/Postal code 200438
Country China
Platform ID GPL25526
Series (1)
GSE244725 Harnessing HetHydrogel: An Approach to Single-Cell Multi-Omics
BioSample SAMN37698652
SRA SRX22004971

Supplementary file Size Download File type/resource
GSM7825047_3T3_percellaverage_sorted.bam 340.6 Mb (ftp)(http) BAM
SRA Run SelectorHelp
Raw data are available in SRA

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