Lysis buffer

RNA was isolated from 5,000 to 10,000 DC previously facs-sorted into 40 μl of lysis buffer (Life Technologies). Dynabeads mRNA Direct Purification Kit (Life Technologies) was used following manufacturer's guidelines. RNA was reverse transcribed with High Capacity cDNA Transcription Kit (Applied Biosystems). PCR were performed with Platinum SYBR Green qPCR SuperMix (Life Technologies) and QuantStudio 6 Flex (Applied Biosystems). Quantification of the PCR signals of each sample was performed by comparing the cycle threshold values (Ct), in duplicate, of the gene of interest with the Ct values of the TBP housekeeping gene.
Primers used in the research: Il6 Fwd 5′-CCTCTCTGCAAGAGACTTCCAT-3′, Il6 Rev 5′-ACAGGTCTGTTGGGAGTGGT-3′, Il12a (p35) Fwd 5'-GCCACCCTTGCCCTCCTAA-3', Il12a (p35) Rev 5'-GGTTTGGTCCCGTGTGATGTC-3', Irf1 Fwd 5′-GTTGTGCCATGAACTCCCTG-3′, Irf1 Rev 5'-TGGACTTTCTCTCTTTCCTCTGG-3′, Ccr7 Fwd 5′-CTCCTTGTCATTTTCCAGGTGTG-3′, Ccr7 Rev 5′-GGCAGGAACCAGGCCTTAAA-3′, Tbp 5′-GAAGCTGCGGTACAATTCCAG-3′, Tbp Rev 5'-CCCCTTGTACCCTTCACCAAT-3′.

The tissue levels of selected enzymes associated with lipid metabolism were determined according to previous procedures with minor modifications [31 (link)]. Briefly, 1 g tissue sample was pulverized in liquid nitrogen, and then 100 mg pulverized sample was collected and placed into 1 mL ice-cold lysis buffer (Cat #: T2327, Life Technologies Inc.) and homogenized in an ice-water bath. The lysis buffer contained 1% protease inhibitor cocktail (Cat #: P8340, Sigma, St Louis, MO, USA). Following homogenization, samples were centrifuged at 10,000 g for 5 min at 4°C. After removal of the fat cake, the resulting supernatant was collected, and the pellet was re-suspended in 1 mL lysis buffer to repeat the process of homogenization and centrifugation as described above. The two collections of supernatant were combined and used for enzyme assays. The concentrations of total protein, ACACB, FASN and LPL in the supernatant were determined with the same kits and instruments as described for serum analyses.

The neutralization activities of the IgGs and antibody fragments of anti-CD4i mAbs were evaluated using TZM-bl cells, as described previously [19 (link)]. Briefly, serial dilutions of antibody and virus (400 TCID₅₀) were pre-incubated at 37 °C for 1 h in 96-well tissue culture plates. The TZM-bl cell suspension containing DEAE-Dextran (25 µg/ml) in DMEM with 10% FCS was added to each well. After incubation for 48 h at 37 °C with 5% CO₂, the wells were washed once with PBS and lysed with lysis buffer (Life Technologies, Carlsbad, CA, USA). The lysate was transferred to an opaque white plate containing-galactosidase substrate (Life Technologies) and incubated for 1 h with protection from light. The galactosidase activity was measured using a Centro XS3 LB960 luminometer (Berthold Technologies, Bad Wildbad, Germany). The reduction in infectivity was determined by comparing the relative light units (RLU) in the presence and absence of antibody and was expressed as a percentage of neutralization. The half-maximal (50%) inhibitory concentration (IC₅₀) was calculated using nonlinear regression and defined as the concentration that caused a 50% reduction in luciferase activity.

For bulk RNA-seq analysis, 5,000 DC were facs-sorted into 1.7 ml LoBind microtubes (Eppendorf) containing 40 μl of lysis buffer (Life Technologies). RNA was captured with Dynabeads mRNA Direct Purification Kit (Life Technologies) according to the manufacturer's instructions. The RNA-seq protocol for the generation of libraries is a derivation of MARS-seq (36 (link)). RNA-seq libraries were sequenced using Illumina NexSeq-500, raw data were mapped to the genome (NCBI37/mm9) using HISAT (version 0.1.6) (57 (link)), only reads with unique mapping were considered. Gene expression levels were calculated using HOMER software package (analyzeRepeats.pl rna mm9 -d < tagDir> -count exons -condenseGenes -strand + -raw) (58 (link)). Normalization and differential expression analysis were done using the DESeq2 R-package (Bioconductor, https://bioconductor.org/packages/release/bioc/html/DESeq2.html) (59 (link)). Differentially expressed genes were selected using a 2-fold change and p-value < 0.05 (Figures 3B,D), or a 1.8-fold-change and p-value < 0.05 (Figures 4B,C) or p-value < 0.01(Figure 4D) between at least two conditions. Gene expression matrix was clustered using a k-means algorithm (Matlab function kmeans) with correlation as the distance metric. Heat maps were generated using Genee software.

Early Arabidopsis embryo isolation and the separation of apical and BC lineages of early proembryos were performed according to our previous protocol^{11 (link)}. For embryo isolation, Arabidopsis seeds were collected in the enzyme solution (0.1% cellulase R10, 0.08% macerozyme R10, 80 mM d-Sorbitol, 10% glycerol, 0.058% MES, pH = 5.8) for 30 min at 25 °C, followed by three washes in the washing solution (80 mM d-Sorbitol, 10% glycerol, 0.058% MES, pH = 5.8). Early embryos were then isolated manually from the seeds with two fine glass needles under an inverted microscope. Apical and BC lineages of early proembryos were separated using LMD System (Leica, Germany), and collected under an inverted microscope by the micromanipulation. Isolated apical and BC lineages were extensively washed four times and transferred into lysis buffer (Life Technologies, USA) and stored in liquid nitrogen for mRNA isolation.