GSM3733721: MLN_RFP_neg_3: RNA-seq in MLN T-bet- cDC2, rep2; Mus musculus; RNA-Seq
Single cell suspensions of cells were prepared from spleen or mesenteric lymph nodes. Cells were then stained for 30 min on ice in FACS buffer (PBS, 2% FBS, 2 mM EDTA) with antibodies against cell surface proteins. For exclusion of dead cells, samples were stained with Sytox Blue cell viability reagent immediately prior to cell sorting. Stained cells were sorted using a FACSAria II (BD). For RNA-seq, cells were sorted into Trizol LS. RNA-seq: RFP+ (T-bet+) or RFP- (T-bet-) cDC2s or cDC1s were were FACS- sorted from Tbx21RFP-Cre mice directly into Trizol-LS (ThermoFisher) and volume was adjusted post-sort. Samples were stored at −80°C prior to RNA extraction. Total RNA was extracted and assessed for nucleic acid quantity and quality on the Agilent BioAnalyzer. SMARTer amplification was followed by Illumina TruSeq paired- end library preparation following manufacturer's protocols. Samples were sequenced on the Illumina HiSeq 2500 to an average depth of 30 million 50-bp read pairs per sample.  ATAC-seq: FACS-sorted cell populations (50,000 cells) were washed with PBS and cell pellets were resuspended in 50ul of cold lysis buffer followed by centrifugation. Cell pellets were resuspended in 50ul of Tn5 transposase mixture: 1x Tagment DNA Buffer, 2.5ul Tagment DNA Enzyme (Nextera DNA Library Preparation Kit, Illumina). Cells were incubated at 42°C for 45 minutes with agitation followed by DNA isolation using the MinElute Reaction Cleanup Kit (QIAGEN, Hilden, Germany). Construction of ATAC-seq libraries included an initial round of PCR in a total volume of 50uL using the NEBNext High-Fidelity 2X PCR Master Mix (New England Biolabs, MA, USA) with primers (1.25M each) from (Buenrostro et al., 2015) with the following thermal cycles: 5 minutes at 72°C, 30 s at 98°C, followed by 5 cycles [98°C for 10 s, 63°C for 30 s and 72°C for 60 s]. To avoid over amplification of libraries which result in GC bias, 5uL of the PCR-amplified DNA were subjected to qPCR (BioRad Real-Time PCR System) in a volume of 15uL using SYBR Green dye (final 0.6x SYBR Green I, Life Technologies) and with the respective primers (1.25M each). Following qPCR [30 s at 98°C, followed by 30 cycles (98°C for 10 s, 63°C for 30 s and 72°C for 60 s)], amplification curves were analyzed and the optimal number of PCR cycles for each sample were estimated with cycle thresholds reaching 1/3 of the maximum. Upon selecting the cycle threshold, the remaining 45uL of the initial PCR mix was subjected to a second round of PCR with the following thermal cycles: 30s at 98°C, followed by n cycles [98°C for 10 s, 63°C for 30 s and 72°C for 60 s]. The libraries were purified by Agencourt AMPure XP beads (x1.8 vol.). Samples were quantified and prepared for sequencing by the Integrated Genomics Core at Memorial Sloan Kettering Cancer Center.  After PicoGreen quantification and quality control by Agilent BioAnalyzer, libraries were pooled equimolar and run on a HiSeq 4000 in a 50bp/50bp paired end run, using the HiSeq 3000/4000 SBS Kit (Illumina). The average number of read pairs per sample was 122 million.
GEO Accession
GSM3733721