title: Illumina NovaSeq 6000 sequencing; The host & gut microbiome orchestrate smoking cessation-induced weight gain
design: The host & gut microbiome orchestrate smoking cessation-induced weight gain
library_construction_protocol: Mouse treatment: 1) ACG in diet 7500 mg/kg of diet, 2) DMG through osmotic pumps - Alzet osmotic minipumps model 2004 were used (infusing the compound at a rate of 0.25 µl per h for 28 days). The pumps were filled with 200 µl of: DMG to achieve 100mg/kg/day Epididymal adipose tissues from controls and treated (with DMG and ACG) mice (n=4 per group) were dissected and placed in 5 ml of DMEM (w/o phenol red, 4500 mg/l glucose) at room temperature and disintegrated by chopping with sterile scissors. Subsequently 0.2g of collagenase II (Sigma-Aldrich C6885) was added and samples were incubated for 20 minutes at 37C while shaking. 5 ml of cold DMEM was added to each sample and they were filtered through 250 ul mesh. Samples were centrifugated at room temperature for 5 minutes at 300g without breaks and with slowest acceleration. The pellets containing immune cells were recovered and resuspended in ACK buffer (Gibco, A1049201) and incubated for one minute. Then 14 ml of PBS was added and cells were harvested by centrifugation at 500g for 10 minutes. Pellets were resuspended in PBS with 0.04% BSA, filtered through 40um mesh Chromium 3' RNA v3.1 chip (10x Genomics) aiming for 6000 cells per sample.  Libraries were prepared according to manufacturer's instructions