|
| Status |
Public on Jan 14, 2024 |
| Title |
RNA_bM0_2 |
| Sample type |
SRA |
| |
|
| Source name |
blood monocyte derived M0 macrophage
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: blood monocyte derived M0 macrophage
cell type: M0 macrophage
genotype: WT
|
| Treatment protocol |
Stem cell derived monocytes were differentiated from iPSCs using the STEMdiff Monocyte Kit (#05320; STEMCELL Technologies), following the manufacturer recommendations. Monocytes harvested from differentiation culture supernatants were collected via centrifugation at 300xg for 5 minutes and then transitioned to culture in ImmunoCult-SF macrophage medium (STEMCELL Technologies #10961) over a period of several days. We started iPSC-derived monocyte to macrophage differentiation 3 days after monocyte harvest by replacing medium with 100% ImmunoCult-SF macrophage medium containing 10 ng/mL M-CSF (R&D Systems #216-MC-010). To obtain blood-derived monocytes, 50 mL of human blood from apparently healthy anonymous donors was collected on the day of purification. Within 1 hour of blood collection, 12 mL aliquots of blood were diluted 1:1 with Dulbecco’s Phosphate Buffered solution (PBS) (Caisson Labs # PBL02-500ML) + 2% FBS before being loaded onto 15 mL of Lymphoprep (STEMCELL Technologies #07801). The layered blood-Lymphoprep mixture was centrifuged at 800xg for 20 minutes at room temperature with the brake off. After the centrifugation, the upper plasma layer was gently removed and discarded. The buffy coat was collected and washed with PBS + 2% FBS three times with low-speed centrifugation at 180xg for 10 minutes at room temperature. Subsequently, monocytes were purified from the peripheral blood mononuclear cells (PBMCs) via Percoll (Sigma-Aldrich #P4937) gradient centrifugation as previously described. The purified monocytes were plated onto 6-well plates at 2x10E6 cells per well. To differentiate both iPSC-derived and blood-derived monocytes into naïve macrophages, monocytes were plated at 1x10E6 cells per well of a 6-well plate in ImmunoCult-SF macrophage medium supplemented with 10 ng/mL M-CSF and cultured for 4 days. To polarize the naïve macrophages into M1 subtype, the M0 macrophages were incubated in ImmunoCult-SF macrophage medium supplemented with 50 ng/mL Interferon-gamma (R&D Systems #285-IF-100/CF) and 10 ng/mL lipopolysaccharide (Sigma-Aldrich #L2018) for 2 days. To polarize the naïve macrophages into M2 subtype, the M0 macrophages were incubated in ImmunoCult-SF macrophage medium supplemented with 10ng/mL Interleukin-4 (Sigma-Aldrich #H7291) for 2 days.
|
| Growth protocol |
The iPSC line is maintained in mTeSR Plus medium (STEMCELL Technologies #100-0276) using hESC-qualified Matrigel (Corning #CLS354277) coated plates. All cells were maintained at 37C and 5% CO2. All subcultures of iPSC were passaged using 0.5 M EDTA (Invitrogen # AM9260G). Additional culturing conditions for iPSC and blood-derived macrophages are listed in the treatment protocol.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
For RNA-seq, total RNA was harvested using Zymo Quick-RNA Microprep kit (Zymo #R1050), following manufactuere's protocol.
Following manufactuere's protocol, mRNA was enriched and prepared into sequencing library using KAPA mRNA HyperPrep kits (Roche #8098123702). The libraries were sequenced on the NovaSeq 6000 System using 2x151 cycles for a minimum of 20 million pair-end reads per library.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
Sequencing reads were first trimmed for low-quality reads and adapter sequences using BBDuk (BBMap v38.05).
The trimmed reads were mapped to human hg38 genome using HISAT2 v2.1.0.
The number of reads were mapped to human RefSeq exons using FeatureCount (Subread v1.6.2).
The mapped reads were normalized by size factors and analyzed for differential expression using DESeq2 R package v1.34.0.
To visualize relative expression of genes, the fragments per kilobase of transcript per million mapped reads (FPKM) of size-factor normalized read counts were extracted to plot the heatmaps, and the expression level was subsequently scaled to row z-score. The gene ontology enrichment analysis was done using R package clusterProfiler v3.0.4.
Assembly: hg38
Supplementary files format and content: Tab delimited text file including raw counts for each sample
|
| |
|
| Submission date |
Jan 10, 2024 |
| Last update date |
Jan 14, 2024 |
| Contact name |
Sara L Sawyer |
| Organization name |
University of Colorado Boulder
|
| Department |
BioFrontiers Institute
|
| Street address |
3415 Colorado Ave.,
|
| City |
Boulder |
| State/province |
CO |
| ZIP/Postal code |
80309 |
| Country |
USA |
| |
|
| Platform ID |
GPL24676 |
| Series (1) |
| GSE252979 |
Macrophages derived from human induced pluripotent stem cells (iPSCs) serve as a high-fidelity cellular model for investigating HIV-1, dengue, and influenza viruses |
|
| Relations |
| BioSample |
SAMN39399702 |
| SRA |
SRX23158900 |
