Illumina HiSeq 4000 sequencing; scRNA-seq of human fetal lung primary tissues and cell cultures derived from fetal bud tip progenitors under dual SMAD treatment
scRNA-seq of human fetal lung primary tissues and cell cultures derived from fetal bud tip progenitors under dual SMAD treatment
Human tissue research was reviewed and approved by The University of Michigan Institutional Review Board (IRB). Human lung tissue was obtained from the University of Washington Laboratory of Developmental Biology. Tissue was shipped overnight in UW-Belzer's solution on ice. To dissociate human fetal tissue to single cells, tissue was first dissected into regions (trachea/bronchi, small airways, distal lung) using forceps and a scalpel in a petri dish filled with ice-cold 1X HBSS (with Mg2+, Ca2+).  For harvesting cells from the trachea and bronchi, the airways were transferred to a fresh petri dish filled with ice-cold 1X HBSS and were opened longitudinally with spring-loaded microscissors. The epithelium was scraped with a scalpel and a p200 pipette tip, prewashed with 1% BSA in HBSS to reduce cells from sticking, was used to collect epithelial cells and place them in a 15 mL conical tube. For distal lung tissue, a section roughly 1cm2 was cut with a scalpel from the most distal lung regions and minced using a scalpel and forceps. This tissue was then transferred to a 15 mL conical tube.  Dissociation enzymes and reagents from the Neural Tissue Dissociation Kit (Miltenyi, cat. no. 130-092-628) were used, and all incubation steps were carried out in a refrigerated centrifuge pre-chilled to 10ºC unless otherwise stated. All tubes and pipette tips used to handle cell suspensions were pre-washed with 1% BSA in HBSS to prevent adhesion of cells to the plastic. Tissue was treated for 15 minutes at 10ºC with Mix 1 and then incubated for 10 minute increments at 10ºC with Mix 2 interrupted by agitation by pipetting with a P200 pipette until fully dissociated. Cells were filtered through a 70 µm filter coated with 1% BSA in 1X HBSS, spun down at 500g for 5 minutes at 10ºC and resuspended in 500µl 1X HBSS (with Mg2+, Ca2+). 1 mL Red Blood Cell Lysis buffer was then added to the tube and the cell mixture was placed on a rocker for 15 minutes in the cold room (4ºC). Cells were spun down (500g for 5 minutes at 10ºC), and washed twice by suspension in 2 mLs of HBSS + 1% BSA  followed by centrifugation.  Cells were then resuspended in 1% BSA in HBSS with 0.5 units/µL of RNAseI (ThermoFisher cat. no. AM2294) in order to reduce RNA present in the media/buffer. Cells were counted using a hemocytometer (ThermoFisher), then spun down and resuspended (if necessary) to reach a concentration of 700-1000 cells/µL and kept on ice. To dissociate organoids to single cell suspensions, organoids were removed from the Matrigel droplet by vigorous pipetting with a p200 in a small petri dish, then transferred to a 15 mL conical tube containing 8 mL of TrypLE express (ThermoFisher Scientific cat. no. 12605036) and placed in a tissue culture incubator at 37ºC, 5% CO2 on a rocker for no longer than 30 minutes. Every 5 minutes, the tube was removed from the incubator and cells were agitated by pipetting the solution up and down with a P1000 pipette. Cells were confirmed to be at single cell suspension by microscope. After most cells were in a single cell suspension, cells were spun down at 300g for 5 minutes at 4ºC, resuspended in 1% BSA in HBSS, filtered through a 70 µm filter (similar to Fisher Scientific cat. no. 087712) and spun down again at 300g for 5 minutes at 4ºC. All tubes used to handle cell suspensions were pre-washed with 1% BSA in HBSS to prevent adhesion of cells to the plastic walls of tubes. Cells were then resuspended in 100 µL of 1% BSA in HBSS with 0.5 units/µL of RNAseI (ThermoFisher cat. no. AM2294), counted using a hemocytometer (ThermoFisher), then spun down and resuspended (if necessary) to reach a concentration of 700-1000 cells/µL and kept on ice.  For the dual SMAD activation treatment, cultures were treated with 100 ng/mL BMP4 and 100 ng/mL TGFb1, on top of the basal media without the additional growth factors FGF7, CHIR-99021 and ATRA.  Immediately after tissue dissociation, single cell libraries were prepared on the 10x Chromium with a target of 10,000 cells. Chromium Single Cell 3' Library & Gel Bead Kit v2, 16 rxns PN-120237 was used. Immediately after tissue dissociation, single cell libraries were prepared on the 10x Chromium with a target of 10,000 cells. Chromium Single Cell 3' Library & Gel Bead Kit v2, 16 rxns PN-120237 was used.
Experimental Factor: protocol
10x v2
Experimental Factor: stimulus
none
Experimental Factor: time
not applicable
Experimental Factor: organism part
respiratory airway
Experimental Factor: sampling site
middle airway
Experimental Factor: growth condition
in vivo