KEY RESOURCE TABLE
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| HA | BioLegend | Cat# 901501, RRID:AB_2565006 |
| Allophycocyanin (APC)-conjugated secondary antibodies | eBioscience | Cat# 17-4015-82 |
| TfR | DSHB | Cat# G1/221/12, RRID:AB_2201506 |
| FAS | eBioscience | Cat# 14-0959-80, RRID:AB_467396 |
| PVR | BioLegend | Cat# 337602, RRID:AB_2300508 |
| FLAG | Sigma-Aldrich | Cat# F1804, RRID:AB_262044 |
| α-adaptin (α subunit of AP2 adaptor) | BD Biosciences | Cat# 610502, RRID:AB_397868 |
| α-adaptin | Santa Cruz Biotechnology | Cat# SC-17771, RRID:AB_2274034 |
| α-tubulin | eBioscience | Cat# 14-4502-82, RRID:AB_1210456 |
| AAGAB | Bethyl | Cat# A305-593A |
| β2 subunit | Bethyl | Cat# A304-719A, RRID:AB_2620914 |
| μ2 subunit | BD Biosciences | Cat# 611350, RRID:AB_398872 |
| γ-adaptin | Sigma-Aldrich | Cat# A4200, RRID:AB_476720 |
| δ-adaptin | DSHB | Cat# anti-delta SA4, RRID:AB_2056641 |
| Horseradish peroxidase (HRP)-conjugated anti-HA | Roche | Cat# 12013819001, RRID:AB_390917 |
| HRP-conjugated anti-rabbit IgG | Sigma-Aldrich | Cat# A6154, RRID:AB_258284 |
| HRP-conjugated anti-mouse IgG | Sigma-Aldrich | Cat# A6782, RRID:AB_258315 |
| Alexa Flour 488-conjugated anti-rabbit IgG | Thermo Fisher Scientific | Cat# A11008, RRID:AB_142165 |
| Alexa Flour 568-conjugated anti-rabbit IgG | Thermo Fisher Scientific | Cat# A11004, RRID:AB_2534072 |
| Bacterial and Virus Strains | ||
| Electrocompetent E. coli | Lucigen | Cat# 60242 |
| BL21 Gold DE3 competent E. coli | Stratagene | Cat# 230132 |
| Chemicals, Peptides, and Recombinant Proteins | ||
| Accutase | Innovative Cell Technologies | Cat# AT 104 |
| Insulin | Sigma-Aldrich | Cat# I0516 |
| Citifluor AF3 anti-fade solution | Electron Microscopy Sciences | Cat# 17972-25 |
| Dynasore | Sigma-Aldrich | Cat# D7693 |
| FB Essence | VWR | Cat# 10803-034 |
| 3,3’,5-Triiodo-L-thyronine | Sigma-Aldrich | Cat# T2877 |
| Fetal bovine serum (FBS) | Sigma-Aldrich | Cat# F2442 |
| Indomethacin | Sigma-Aldrich | Cat# I7378 |
| ProLong Antifade mountant with DAPI | Thermo Fisher Scientific | Cat# P36931 |
| Protease inhibitor cocktail | Roche | Cat# 05056489001 |
| Dexamethasone | Sigma-Aldrich | Cat# D1756 |
| Isobutylmethylxanthine | Sigma-Aldrich | Cat# I5879 |
| Gibson assembly kit | Synthetic Genomics | Cat# GA1200 |
| Saponin | Sigma-Aldrich | Cat# 47036 |
| FuGENE HD | Promega | Cat# E2311 |
| TRIzol reagent | Thermo Fisher Scientific | Cat# 15596018 |
| Critical Commercial Assays | ||
| Genomic DNA isolation kit | Thermo Fisher Scientific | Cat# K0721 |
| Site-directed mutagenesis kit | Agilent | Cat# 210518 |
| Zymo RNA Clean and Concentrator columns | Zymo Research | Cat# R1018 |
| Superscript IV first strand synthesis system | Thermo Fisher Scientific | Cat# 18091050 |
| Sso Advanced SYBER Green Supermix | Bio-Rad | Cat# 1725270 |
| Experimental Models: Cell Lines | ||
| HeLa cells | ATCC | CCL-2 |
| 293T cells | ATCC | CRL-3216 |
| Mouse preadipocytes: inguinal white adipose tissues | Dr. Shingo Kajimura | N/A |
| Oligonucleotides | ||
| Sequencing adaptors, see table S5. | Sanjana et al. (2014) | N/A |
| Sequencing primers, see table S5. | Sanjana et al. (2014) | N/A |
| Secondary library amplification primers, see table S5. | Sanjana et al. (2014) | N/A |
| Secondary library oligos, see table S3. | This paper | N/A |
| CRISPR guide sequence primers, see table S5. | This paper | N/A |
| AP2A2 qPCR primers, see table S5. | This paper | N/A |
| GAPDH qPCR primers, see table S5. | Dr. Roy Parker | N/A |
| Recombinant DNA | ||
| Plasmid: pET-SUMO-humanAAGAB | This paper | N/A |
| Plasmid: pGEX4T-3-mouseAP2A2 | This paper | N/A |
| Plasmid: pACYC-Duet-ratAP2S1 | This paper | N/A |
| Plasmid: p3×FLAG-humanAAGAB | This paper | N/A |
| Plasmid: pcDNA3.1-mouseAP2A2-HA | This paper | N/A |
| Plasmid: pcDNA3.1-ratAP2S1-HA | This paper | N/A |
| Plasmid: pcDNA3.1-humanAP2B1-HA | This paper | N/A |
| Plasmid: pcDNA3.1-mouseAP2M1-HA | This paper | N/A |
| Plasmid: pcDNA3.1-mouseAP2A2 | This paper | N/A |
| Plasmid: pCMV-ratAP2S1-3×FLAG | This paper | N/A |
| Plasmid: pEGFP-N1-humanAAGAB | This paper | N/A |
| Plasmid: pCSII-humanAAGAB | This paper | N/A |
| Plasmid: p3×FLAG-CMV™-14 | Sigma-Aldrich | Cat# E7908 |
| Plasmid: p3×FLAG-CMV™-7.1 | Sigma-Aldrich | Cat# E7533 |
| Plasmid: pcDNA™3.1 (+) | Thermo Fisher Scientific | Cat# V79020 |
| Plasmid: pLenti-HA-GLUT4-GFP | (Muretta et al., 2008) | N/A |
| Plasmid: pLentiCRISPRv2 | (Sanjana et al., 2014) | Addgene: 52961 |
| Plasmid: pLenti-Guide-Hygro | (Gulbranson et al., 2017) | N/A |
| Plasmid: pLenti-CRISPR | (Shalem et al., 2014) | Addgene: 49535 |
| Plasmid: pAdVAntage | Promega | Cat# E1711 |
| Plasmid: pCMV-VSVG | (Stewart et al., 2003) | Addgene: 8454 |
| Plasmid: psPAX2 | Dr. Diderio Trono | Addgene: 12260 |
| Plasmid: pLenti-Guide-Puro | (Sanjana et al., 2014) | Addgene: 52963 |
| Plasmid: pAP1G1 (1-595)-GST | (Ren et al., 2013) | |
| Plasmid: pmCherry-α-adaptin | Dr. Juan Bonifacino | |
| Software and Algorithms | ||
| FASTX-toolkit | Hannon lab website | http://hannonlab.cshl.edu/fastx_toolkit/ |
| MAGeCK | (Li et al., 2014) | https://sourceforge.net/p/mageck/wiki/Home/ |
| EXCEL | Microsoft | N/A |
| KaleidaGraph | Synergy Software | http://www.synergy.com/wordpress_650164087/ |
| Prism (version 7) | GraphPad | https://www.graphpad.com/scientific-software/prism/ |
| R studio | R-project | N/A |
| FlowJo (version 8 and 9) | FlowJo | N/A |