USR_template_replicates
USR_template_replicates
Total RNA was extracted using the PureLink RNA Mini Kit  (Life Technologies), according to the manufacturer's protocol. Concentration was measured on a Nanodrop 2000c Spectrophotometer and RNA integrity and concentration checked on an 2100 Bioanalyzer (Agilent technologies). Isolated ASC RNA from the nine mice was homogenously pooled, aliquoted and frozen together with the RNA from the single mouse at -80  degrees C. Next, cDNA was prepared with half of the total 9M RNA and total 1M RNA using Maxima Reverse Transcriptase (Fermentas) and following the manufacturerÕs protocol. For each individual cDNA reaction 500 ng RNA was used, cDNA from multiple reactions were pooled and stored at -80  degrees C. PCR amplification of the VH IgG genes was performed with a set of 19 forward primers binding to framework 1 of the VDJ regions as previously described 43 and an IgG-specific reverse primer binding to the constant region (5Õ CARK GGA TRR RCH GAT GGGG 3Õ). The Illumina TruSeq Universal Adaptor constituted the 5Õ portion of the forward primers, while the IgG-reverse primer contained the Illumina index primer, thereby directly adding Illumina adapter sequences to PCR products (Figure 7XX). As each cell population (1M and 9M) was sequenced in triplicates, each PCR sample was prepared with its own unique index primer. Analogously to Reddy and colleagues 4, each 50 ul PCR reaction consisted of 0.2 uM of forward primer mix and reverse primer, 5 ul of Thermopol reaction buffer (NEB), 200 uM dNTPs, 2 ul of unpurified cDNA, 0.25 ul Taq DNA polymerase (NEB) filled up with double-distilled water. For each separate indexed sample 10 PCR reactions were run in parallel as follows: 95  degrees C for 3 min; 4 cycles (95  degrees C for 30 sec, 50  degrees C for 30 sec, 68  degrees C for 1 min); 4 cycles (95  degrees C for 30 sec, 55  degrees C for 30 sec, 68  degrees C for 1 min); 20 cycles (95  degrees C for 30 sec, 63  degrees C for 30 sec, 68  degrees C for 1 min); 68  degrees C for 7 min; 4  degrees C storage. PCR clean-up was performed in order to reduce the volume and products were run on a 1 % agarose gel for purification. Bands of ~ 550 bp were gel-excised, purified and prior to sequencing were submitted for a final quality control step on an Bioanalyzer 2100 (Agilent).
Experimental Factor: protocol
pooling of tissues from 9 immunised mice