Stempro accutase cell dissociation reagent

Mammalian cell display vector harboring Fc libraries was transfected into HEK293T cells, together with pMDLg/pRRE, pRSV-Rev and pCMV-VSV-G packaging plasmids, using polyethylenimine (PEI), to produce lentivirus library. The lentivirus was concentrated with Lentivirus Concentration Reagent (BIOMIGA), and the titer was measured with Lenti-X™ p24 Rapid Titer Kit (Takara Bio Inc.).
1×10⁷ HEK293T cells were infected by lentivirus library at low MOI, followed by 48 h of culture to display Fc variants on the cell surface. The cells were then detached by StemPro Accutase Cell Dissociation Reagent (Thermo Fisher Scientific), resuspended in 1 mL blocking buffer (PBS supplemented with 5% FBS (V/V), 1% glucose (W/V), 1 mM Na₂EDTA, 1 mM HEPES) and incubated for 30 min at 4 °C with gentle agitation. Then the cells were incubated with 250 nM (125 nM for the 2^nd and 3^rd rounds of sorting) biotinylated FcγRs (bio-FcγRs) in blocking buffer at 4 °C for 30 min. After washing three times with ice-cold FACS buffer (PBS supplemented with 1% glucose (W/V), 1 mM Na₂EDTA, 1 mM HEPES), the cells were resuspended in 1 mL of blocking buffer containing streptavidin-PE (1:1000 dilution, Thermo Fishier Scientific) and ANTI-FLAG® M2-FITC (1:200 dilution, SIGMA) and incubated at 4 °C for 30 min. The cells were finally washed three times and resuspended in ice-cold FACS buffer for sorting (FACSAria III, BD).

Gingival tissues were obtained as remnants of discarded tissues under the approved Institutional Review Board protocol at the University of Pennsylvania. The isolation and characterization of human gingival tissue‐derived MSCs were described previously 41. To induce NPC‐like cells from GMSCs, adherent monolayer GMSCs were trypsinized and seeded onto poly‐l‐ornithine/laminin‐treated 6‐well plates (2 × 10⁵ cells/well) and cultured with complete α‐minimum essential medium for 24 hours. The medium was replaced with neural cell culture medium (neurobasal medium supplemented with 1% N‐2 Supplement, 1% B27, 100 U/ml penicillin, 100 μg/ml streptomycin, and 550 μM 2‐mercaptoethanol; all from Thermo Fisher Scientific Life Sciences, Waltham, MA, http://www.thermofisher.com), 20 ng/ml epidermal growth factor (EGF) and 20 ng/ml basic fibroblast growth factor (bFGF) (PeproTech, Rocky Hill, NJ, http://www.peprotech.com). Under this condition, neurosphere formation was observed at approximately 3–6 days. The neurospheres were completely dissociated into single cells using StemPro Accutase Cell Dissociation Reagent (Thermo Fisher), and cell viability was determined by Trypan blue staining. Dissociated cells were seeded onto polyornithine/laminin‐precoated dishes for further culture.

Importation of the HES3 and subsequent experiments using hPSCs were authorised by the Robert-Koch Institute (Berlin, Germany) under license number AZ 3.04.0210083. The hPSCs were maintained as undifferentiated colonies on Corning^® Matrigel^® hESC-Qualified Matrix (Corning GmbH, Kaiserslautern, Germany) coated plates in StemMACS™ iPS-Brew XF media (Milteny Biotech, Bergish Gladbach, Germany) supplemented with 50 U/mL penicillin, and 50 U/mL streptomycin (Thermo Fisher, Waltham, MA, USA) at 37 °C and 5% CO₂. Medium was changed every other day. When confluent, the hPSC colonies were dissociated into single cells using StemPro^® Accutase^® Cell Dissociation Reagent (Thermo Fisher, Waltham, MA, USA) and plated onto Matrigel-coated 60mm plates (Corning GmbH, Kaiserlautern, Germany).

We followed the HiChIP protocol published by Mumbach et al.^{37 (link)}, using antibody to H3K27ac (Abcam, ab4729) with the following modifications. The EBs were first treated with StemPro Accutase Cell Dissociation Reagent (Thermo Fisher) at 37 °C for 10–15 min with pipetting. Approximately one million cells were crosslinked with freshly prepared 1% formaldehyde. The pellet was then resuspended in 500 μl of ice-cold Hi-C Lysis buffer. After digestion with 25 U (5 μl of 5U/μl) MboI restriction enzyme and ligation, the nuclear pellet was brought up to 880 μl of Nuclear Lysis Buffer. Samples were sheared using a Covaris E220 using the following parameters: fill level = 10, duty cycle = 5, PIP = 140, cycles/burst = 200, time = 2 min and then clarified by centrifugation for 15 min at 16,100 × g at 4 °C. The samples were precleared with 6 μl Dynabeads Protein A (Thermo Fisher) at 4 °C for 1 h. We then added 2.5 μg of antibody to H3K27ac, and captured the chromatin-antibody complex with 6 μl of Dynabeads Protein A. Approximately 2–4 ng of ChIP DNA was obtained following Qubit quantification. The amount of Tn5 used and number of PCR cycles performed were based on the post-ChIP Qubit amounts, as described in the HiChIP protocol^{37 (link)}. The library was sequenced on Illumina NextSeq 500 with 75 bp paired-end reads. Total 13 million cells were used in HiChIP experiment.