Human-induced pluripotent stem cells (hiPSCs) (https://www.hipsci.org/lines/#/lines/HPSI0714i-nufh_3 , https://www.hipsci.org/lines/#/lines/HPSI0914i-euts_1 )59 (link) were thawed and cultured under feeder-free conditions on Vitronectin XF™-coated (Stem Cell, #07180) 10 cm tissue culture treated plates (Corning) in complete Essential 8 medium (Life Technologies #A1517001) supplemented with 1% Penicillin/Streptomycin (Invitrogen, #15140122). Cells were incubated at 37°C, 5% CO2 and media changed every day except for the day of passaging. Cells were routinely passaged using 0.5 mM EDTA (Life Technologies #AM9260G) at least 3 times post-thawing before collection. HiPSCs were harvested using Accutase (Millipore, #SCR005) to generate a single cell suspension. Single cells were resuspended in 1X PBS (Thermo Fisher Scientific, #10010023) and passed through a 40uM filter (Corning #CLS431750-50EA) before FAC sorting (BD Influx™ Cell Sorter) into 96-well plates (Framestar, #4TI-0960) containing 2 μl lysis buffer with 1 U/μl Rnase Inhibitor (RRI) and ERCC spike in mix at a 1:20 dilution (Ambion Cat #4456740). Plates were promptly sealed (Thermo Fisher Scientific #AB0626), spun down and frozen at -80 °C until further processing.
Ab0626
Manufactured by Thermo Fisher Scientific
The AB0626 is a laboratory equipment product offered by Thermo Fisher Scientific. It is designed to perform a core function within the laboratory setting. However, a detailed and unbiased description of its specific features and capabilities cannot be provided while maintaining the requested conciseness and lack of interpretation or extrapolation.
Automatically generated - may contain errors
Lab products found in correlation
Vitronectin xf coated, by STEMCELL (3 mentions)
Am9260g, by Thermo Fisher Scientific (3 mentions)
Accutase, by Merck Group (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Cls431750 50ea, by BD (3 mentions)
Ab1384, by Thermo Fisher Scientific (1 mentions)
Triethylammonium bicarbonate buffer, by Merck Group (1 mentions)
Bravo pipetting robot, by Agilent Technologies (1 mentions)
Speedvac, by Eppendorf (1 mentions)
5 protocols using ab0626
1
Single-cell RNA-seq of hiPSCs
2
Single-cell Proteomics Sample Preparation
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Cells were harvested as single-cell suspension and prepared for MS analysis using Nano-ProteOmic sample Preparation (nPOP) as described by Leduc et al.50 ,51 . The automated collection of prepared samples had not been developed yet26 (link) and so samples were manually collected using a pipette (using 5μl of mass spectrometry grade Acetonitrile then water respectively) and transferred into a 384-well plate (Thermo AB1384). The samples were then dried down in a SpeedVac vacuum evaporator and resuspended in 1.07μl of 0.1% Formic Acid (buffer A) and tightly sealed using an aluminium foil cover (Thermo Fisher AB0626).
For the bulk experiments, cells were harvested (in MS grade water, at roughly 2000 cell/μl) and frozen at −80C. The samples were prepared using mPoP52 , following guidelines for the digest of carriers as outlined in Petelski et al.30 (link). Post digest, the samples were dried down in a SpeedVac vacuum evaporator and resuspended at a concentration of 1 μg/μl in 0.1% Formic Acid (buffer A) in a glass insert with polyspring within an HPLC vial.
For the bulk experiments, cells were harvested (in MS grade water, at roughly 2000 cell/μl) and frozen at −80C. The samples were prepared using mPoP52 , following guidelines for the digest of carriers as outlined in Petelski et al.30 (link). Post digest, the samples were dried down in a SpeedVac vacuum evaporator and resuspended at a concentration of 1 μg/μl in 0.1% Formic Acid (buffer A) in a glass insert with polyspring within an HPLC vial.
3
Single-cell RNA-seq of hiPSCs
4
Automated Peptide Preparation and Dimethylation
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Peptides were prepared semi-automated on a Bravo pipetting robot (Agilent), similarly to as described previously11 . During each incubation step, plates were tightly sealed with two stacked aluminum lids to avoid evaporation (Thermo Fisher Scientific, AB0626). For this, plates were removed from the freezer and centrifuged. The wells were then washed on the robot with 28 µl of 100% ACN and dried in a SpeedVac (Eppendorf) at 45 °C for 20 min. Shapes were then resuspended in 6 µl of 60 mM triethylammonium bicarbonate buffer (pH 8.5, Sigma) with 0.013% DDM (Sigma), and cooked for 30 min at 95 °C in a PCR cycler at a lid temperature of 110 °C. After addition of 1 µl of 80% ACN (final concentration 10%), samples were incubated for another 30 min at 75 °C, cooled briefly, and 1 µl with 4 ng LysC and 6 ng trypsin was added. The samples were digested for 18 h, and then 1 µl of either intermediate (CD2O) or heavy formaldehyde (13CD2O) was added to a final concentration of 0.15%. Without delay, either light (NaBH3CN) or heavy (NaBD3CN) sodium cyanoborohydrate were added to 0.023 M to retrieve Δ4 and Δ8 dimethyl-labeled single-shape samples. The sealed plate was then incubated at room temperature for 1 h, and the reaction was quenched to 0.13% ammonia and acidified to 1% TFA.
5
Feeder-Free Culture of hiPSCs
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Human-induced pluripotent stem cells (hiPSCs) were thawed and cultured under feeder-free conditions on Vitronectin XF™-coated (Stem Cell, #07180) 10 cm tissue culture treated plates (Corning) in complete Essential 8 medium (Life Technologies #A1517001) supplemented with 1% Penicillin/Streptomycin (Invitrogen, #15140122). Cells were incubated at 37°C, 5% CO2 and media changed every day except for the day of passaging. Cells were routinely passaged using 0.5 mM EDTA (Life Technologies #AM9260G) at least 3 times post-thawing before collection. HiPSCs were harvested using Accutase (Millipore, #SCR005) to generate a single cell suspension. Single cells were resuspended in 1X PBS (Thermo Fisher Scientific, #10010023) and passed through a 40uM filter (Corning #CLS431750-50EA) before FAC sorting (BD Influx™ Cell Sorter) into 96-well plates (Framestar, #4TI-0960) containing 2µl lysis buffer with 1U/µl Rnase Inhibitor (RRI) and ERCC spike in mix at a 1:20 dilution (Ambion Cat #4456740). Plates were promptly sealed (Thermo Fisher Scientific #AB0626), spun down and frozen at -80 °C until further processing.
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