NextSeq 500 sequencing; Massively parallel reporter assays (MPRAs) of 175bp human genomic fragments containing variants in high LD with lead variants from obesity GWAS associations
Massively parallel reporter assays (MPRAs) of 175bp human genomic fragments containing variants in high LD with lead variants from obesity GWAS associations
HT22: Cells were maintained in DMEM (Gibco # 11995-065) supplemented with 10% FBS and 5% penicillin-streptomycin solution in 5% CO2. We plated 250K cells into wells of 6 well plates and transfected one day later with Lipofectamine LTX & Plus reagent (Invitrogen; #15338100) when 60-70% confluent. 3T3-L1: Cells were maintained in DMEM (Gibco # 11995-065) supplemented with 10% FBS, 5% penicillin-streptomycin solution, 0.8mg/ml Biotin (Sigma; #B4639) and 0.8mg/ml Panthotenic Acid in 5% CO2. We plated 20k cells into wells of 6 well plates and transfected 2.5 days later with Lipofectamine LTX & Plus reagent (Invitrogen; #15338100) when 30-50% confluent. GT1-7: Cells were maintained in High Glucose DMEM (Gibco # 10313-021) supplemented with 10% FBS, 5% penicillin-streptomycin solution and 1X Glutamax in 5% CO2. We plated 750k cells into wells of 6 well plates and transfected the next day with Lipofectamine LTX & Plus reagent (Invitrogen; #15338100) when 60-70% confluent. SGBS Preadipocyte (D0): 30,000 SGBS preadipocyte cells were plated into 24 well plates and transfected with Polyplus jetPEI DNA transfection reagent (Polyplus; #101-10N) when 50% confluent. These cells were collected 48 hours later for RNA processing. SGBS Adipocyte (D8): Cells were plated as described above for differentiation. On differentiation day 8, cells were transfected with Lipofectamine LTX & Plus reagent (Invitrogen; #15338100) and collected on differentiation day 10.  mRNA was converted to cDNA using Superscript III Reverse Transcriptase. cDNA was treated with RNAse A (invitrogen #12091-021) and RNAse T1 (ThermoFisher #EN0541) for one hour and then cleaned with the Qiagen Minelute PCR purification kit. After transfection, cells were lysed using a 20g needle and RNA was extracted using Qiagen RNeasy mini kit.  Libraries were amplified with 10-11 PCR cycles using Q5 Hot Start High-Fidelity 2x Master Mix (NEB #M0494S) and pooled before clean up using Agencourt AMPure XP beads (0.6x + 1.2x double cleanup; Beckman Coulter; #A63882).