Instrument: BGISEQ-500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: White adipose tissue (epididyma fat) were collected from mice, flash frozen on liquid nitrogen, and total RNA was harvested using Trizol reagent RNA was purified using the poly-A containing mRNA molecules using poly-T oligo-attached magnetic beads. Following purification,the RNA is fragmented into small pieces using divalent cations under elevated temperature.The cleaved RNA fragments are copied into first strand cDNA using reverset ranscriptase and random primers.This is followed by second strand cDNA synthesis using DNA PolymeraseI and RNaseH. These cDNA fragments then have the addition of a single 'A' base and subsequent ligation of the adapter.The products are then purifiedand enriched with PCR amplificat ion.We then quant ified the PCR yield by Qubit and pooled samples together to make a single strand DNA circle (ssDNAcircle), which gave the final library.DNA nanoballs(DNBs)were generated with the ssDNAcircle 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 100bp were read through on the BGISEQ-500platform for the following data analysis study.For this step, the BGISEQ-500 platform combines the DNAnanoball-based nanoarrays and stepwise sequencing using Combinational Probe-Anchor Synthesis Sequencing Method.