Cln1R151X, Cln2R207X and wild type mice, fecal samples at 3 months of age, 16S rRNA V4 sequencing
A modern version of 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) modified for the Illumina MiSeq platform was used to determine the microbiota composition in the mouse fecal samples. This method has been used in many studies to analyze the microbiota in different species (cattle, mice, pigs and humans) and environmental samples [see e.g. (Schaible et al. 2011; Sun et al. 2011; Wang et al. 2016)]. PCR primers 515F (GTGYCAGCMGCCGCGGTAA) and 806R (GGACTACNVGGGTWTCTAAT) for the 16S rRNA gene V4 variable region with barcode on the forward primer were used in a 30 cycle PCR (5 cycle used on PCR products) using the HotStarTaq Plus Master Mix Kit (Qiagen, Valencia, CA) and the following conditions: 94C for 3 min, then 28 cycles of 94C for 30 s, 53C for 40 s and 72C for 1 min, followed by a final elongation step at 72C for 5 min. Following amplification, PCR products were run in a 2% agarose gel to examine the success of amplification and the relative intensity of bands. Multiple samples were pooled together (e.g., 100 samples) in equal proportions based on their molecular weight and DNA concentrations. To purify the pooled samples calibrated Ampure XP beads were used. The pooled and purified PCR products were used to prepare DNA library in accordance with the Illumina TruSeq DNA library preparation protocol. An Illumina MiSeq system was used for sequencing according to Illuminas guidelines. #SampleID BarcodeSequence LinkerPrimerSequence BarcodeName Repeats ProjectName Description 8844.Cln2R207xWT GATCGATC GTGCCAGCMGCCGCGGTAA 515Fbar31 031616AK515F 8844.Cln2R207xWT 8845.Cln2R207xWT GATCGTAC GTGCCAGCMGCCGCGGTAA 515Fbar32 031616AK515F 8845.Cln2R207xWT 8846.Cln2R207xWT GATCGTTG GTGCCAGCMGCCGCGGTAA 515Fbar33 031616AK515F 8846.Cln2R207xWT 8847.Cln2R207xWT GATCTCCT GTGCCAGCMGCCGCGGTAA 515Fbar34 031616AK515F 8847.Cln2R207xWT 8848.Cln2R207xWT GATCTCGA GTGCCAGCMGCCGCGGTAA 515Fbar35 031616AK515F 8848.Cln2R207xWT 8794.6J.WT GATCTGCA GTGCCAGCMGCCGCGGTAA 515Fbar36 031616AK515F 8794.6J.WT 8795.6J.WT GATCTGGT GTGCCAGCMGCCGCGGTAA 515Fbar37 031616AK515F 8795.6J.WT 8796.6J.WT GATGACCT GTGCCAGCMGCCGCGGTAA 515Fbar38 031616AK515F 8796.6J.WT 8797.6J.WT GATGACGA GTGCCAGCMGCCGCGGTAA 515Fbar39 031616AK515F 8797.6J.WT 9092.6J.WT GATGAGCA GTGCCAGCMGCCGCGGTAA 515Fbar40 031616AK515F 9092.6J.WT 8786.Cln1R151x.mixed.KO GATGAGGT GTGCCAGCMGCCGCGGTAA 515Fbar41 031616AK515F 8786.Cln1R151x.mixed.KO 8980.Cln1R151x.mixed.KO GATGCAAG GTGCCAGCMGCCGCGGTAA 515Fbar42 031616AK515F 8980.Cln1R151x.mixed.KO 8981.Cln1R151x.mixed.KO GATGCATC GTGCCAGCMGCCGCGGTAA 515Fbar43 031616AK515F 8981.Cln1R151x.mixed.KO 8982.Cln1R151x.mixed.KO GATGCTAC GTGCCAGCMGCCGCGGTAA 515Fbar44 031616AK515F 8982.Cln1R151x.mixed.KO 8983.Cln1R151x.mixed.KO GATGCTTG GTGCCAGCMGCCGCGGTAA 515Fbar45 031616AK515F 8983.Cln1R151x.mixed.KO 8940.Cln1R151x.KO GATGGAAC GTGCCAGCMGCCGCGGTAA 515Fbar46 031616AK515F 8940.Cln1R151x.KO 8972.Cln1R151x.KO GATGGATG GTGCCAGCMGCCGCGGTAA 515Fbar47 031616AK515F 8972.Cln1R151x.KO 8973.Cln1R151x.KO GATGGTAG GTGCCAGCMGCCGCGGTAA 515Fbar48 031616AK515F 8973.Cln1R151x.KO 8986.Cln1R151x.KO GATGGTTC GTGCCAGCMGCCGCGGTAA 515Fbar49 031616AK515F 8986.Cln1R151x.KO 8987.Cln1R151x.KO GATGTCCA GTGCCAGCMGCCGCGGTAA 515Fbar50 031616AK515F 8987.Cln1R151x.KO 8737.Cln2R207x.mixed.KO GATGTCGT GTGCCAGCMGCCGCGGTAA 515Fbar51 031616AK515F 8737.Cln2R207x.mixed.KO 8738.Cln2R207x.mixed.KO CCTAGGCA GTGCCAGCMGCCGCGGTAA 515Fbar52 031616AK515F 8738.Cln2R207x.mixed.KO 8739.Cln2R207x.mixed.KO CCTAGGTA GTGCCAGCMGCCGCGGTAA 515Fbar53 031616AK515F 8739.Cln2R207x.mixed.KO 8740.Cln2R207x.mixed.KO CCTATAGC GTGCCAGCMGCCGCGGTAA 515Fbar54 031616AK515F 8740.Cln2R207x.mixed.KO 8750.Cln2R207x.mixed.KO CCTCACGT GTGCCAGCMGCCGCGGTAA 515Fbar55 031616AK515F 8750.Cln2R207x.mixed.KO
SUB7283983
031616AK515F