Memory-seq analysis of TRL4 signalling in macrophages
Memory-seq analysis of TRL4 signalling in macrophages
After treatment, cells were harvested by scraping and stored in RNAlater (Invitrogen AM7020) for stable storage. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) high glucose with sodium pyruvate and sodium bicarbonate (Sigma-Aldrich D6429) supplemented with 10% heat inactivated foetal bovine serum (FBS; Gibco 10500064). All incubations were done at 37oC 5% CO2 and all cell handling under laminar air flow unless otherwise stated. Fluorescence activated cell sorting (FACS) was used to sort single iBMDMs into prewarmed DMEM+FBS in 96 well TC treated plates and incubated to generate clonal populations. To reduce clumping during growth from single cells, cells were dispersed by gently reverse pipetting on days 5 and 8. When cells were 60-80% confluent (day 12-14) clonal populations were transferred to 6 well TC treated plates for an additional 24h growth. Mixed populations were seeded directly into TC treated 6 well plates at a similar density to clonal populations. Lipid A diphosphoryl from Salmonella enterica serotype Minnesota Re595 (Sigma-Aldrich L0774) stock was prepared at 500mg ml-1 in 50% DMSO and stored at -20oC until use. Clonal or mixed populations in 6 well plates at approximately 50% confluence were stimulated with Lipid A at a final concentration of 500ng ml-1 for 3h. Unstimulated controls were treated with an equivalent volume of 50% DMSO for 3h. Cells were pelleted by centrifugation and RNAlater removed prior to RNA extraction with the RNeasy Plus mini kit (Qiagen 74136) according to the manufacturer's instructions. RNA was stored at -80oC. RNA concentration, purity and integrity was assessed by Quant-it Qubit RNA BR assay (Invitrogen 10266793), nanodrop and RNA ScreenTape Tapestation analysis (Agilent 5067-5577 and 5067-5576) respectively. Quality and integrity of the RNA samples were assessed using a 4200 TapeStation (Agilent Technologies) and then libraries generated using the Illumina® Stranded mRNA Prep. Ligation kit (Illumina, Inc.) according to the manufacturer's protocol.