title: WT RNA seq of mouse lungs challenged with House Dust Mite
design: Whole mouse lungs were frozen in RNAlater. Whole lung RNA was extracted using RNAeasy kit (Qiagen, Germany) according to the manufacturer's instructions. We used Agilent 2100 Bio analyzer (Agilent RNA 6000 Nano Kit) to do the total RNA sample QC: RNA concentration, RIN value,28S/18S and the fragment length distribution. The first step in the workflow involved purifying the poly-A containing mRNA molecules using poly-T oligo attached magnetic beads. Following purification, the mRNA was fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. This was followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments had addition of a single 'A' base and subsequent ligation of the adapter. The products were then purified and enriched with PCR amplification. PCR yields were quantified by Qubit and pooled samples together to make a single strand DNA circle (ssDNA circle), which gave the final library. DNA nanoballs (DNBs) were generated with the ssDNA circle by rolling circle replication (RCR) to enlarge the fluorescent signals at the sequencing process. The DNBs were loaded into the patterned nanoarrays and pair-end reads of 100 bp were read through on the DNBseq platform for the following data analysis study. For this step, the DNBseq platform combines the DNA nanoball-based nanoarrays and stepwise sequencing using Combinational Probe-Anchor Synthesis Sequencing Method.