title: Illumina HiSeq 4000 sequencing; Massively parallel reporter assays (MPRAs) of 175bp human genomic fragments containing variants in high LD with lead variants from obesity GWAS associations.
design: Massively parallel reporter assays (MPRAs) of 175bp human genomic fragments containing variants in high LD with lead variants from obesity GWAS associations.
library_construction_protocol: 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).