title: GSM7676068: Unbalanced Diet 24 9 weeks; rat gut metagenome; OTHER
library_construction_protocol: On the sacrifice day, caecum from all rats was collected and stored at -80°C. 0.25 gr of defrosted caecum content was extracted using the QIAamp PowerFecal kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The DNA extraction was stored at -80°c until sent in dry ice to the University of Illinois in Chicago for 16S rRNA sequencing of the V4 region using 515F–806R primers. Amplicons were generated using a two-stage PCR amplification protocol as described previously (Naqib et al. (2018)). The primers contained 5' common sequence tags (known as common sequence 1 and 2, CS1 and CS2) as described previously (e.g. Moonsamy et al. 2013). First stage PCR amplifications were performed in 10 microliter reactions in 96-well plates, using MyTaq HS 2X mastermix (Bioline). PCR conditions were 95°C for 5 minutes, followed by 28 cycles of 95°C for 30”, 55°C for 45” and 72°C for 60”. Subsequently, a second PCR amplification was performed in 10 microliter reactions in 96-well plates. A mastermix for the entire plate was made using MyTaq HS 2X mastermix. Each well received a separate primer pair with a unique 10-base barcode, obtained from the Access Array Barcode Library for Illumina (Fluidigm, South San Francisco, CA; Item# 100-4876). These AccessArray primers contained the CS1 and CS2 linkers at the 3' ends of the oligonucleotides. Cycling conditions were: 95°C for 5 minutes, followed by 8 cycles of 95°C for 30”, 60°C for 30” and 72°C for 30”. Libraries were then pooled in equal volume and the pool was purified using an AMPure XP cleanup protocol (0.6X, vol/vol; Agencourt, Beckmann-Coulter) to remove fragments smaller than 300 bp.