Cln3-/- and wild type mice, tap water vs. acidified drinking water, fecal samples at 3 and 6 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. 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 Illumina's guidelines. #SampleID BarcodeSequence LinkerPrimerSequence BarcodeName Repeats ProjectName Description 1.129S6.WT.Tap.2043.2.3m GAGTCTGT GTGCCAGCMGCCGCGGTAA 515Fbar13 102115AK515F 1.129S6.WT.Tap.2043.2.3m 2.129S6.WT.Tap,2131.3.3m GAGTGACA GTGCCAGCMGCCGCGGTAA 515Fbar14 102115AK515F 2.129S6.WT.Tap,2131.3.3m 3.129S6.WT.Tap.2480.1.3m GAGTGAGT GTGCCAGCMGCCGCGGTAA 515Fbar15 102115AK515F 3.129S6.WT.Tap.2480.1.3m 4.129S6.WT.Tap.2480.2.3m GAGTGTCT GTGCCAGCMGCCGCGGTAA 515Fbar16 102115AK515F 4.129S6.WT.Tap.2480.2.3m 5.129S6.WT.Tao.2481.1.3m GAGTGTGA GTGCCAGCMGCCGCGGTAA 515Fbar17 102115AK515F 5.129S6.WT.Tao.2481.1.3m 6.129S6.WT.Tap.2043.2.6m GAGTTCAC GTGCCAGCMGCCGCGGTAA 515Fbar18 102115AK515F 6.129S6.WT.Tap.2043.2.6m 7.129S6.WT.Tap.2131.3.6m GAGTTCTG GTGCCAGCMGCCGCGGTAA 515Fbar19 102115AK515F 7.129S6.WT.Tap.2131.3.6m 8.129S6.WT.Tap.2480.1.6m GAGTTGAG GTGCCAGCMGCCGCGGTAA 515Fbar20 102115AK515F 8.129S6.WT.Tap.2480.1.6m 9.129S6.WT.Tap.2480.2.6m GAGTTGTC GTGCCAGCMGCCGCGGTAA 515Fbar21 102115AK515F 9.129S6.WT.Tap.2480.2.6m 10.129S6.WT.Tap.2481.1.6m GATCACCA GTGCCAGCMGCCGCGGTAA 515Fbar22 102115AK515F 10.129S6.WT.Tap.2481.1.6m 11.129S6.WT.Acidified.2041.2.3m GATCACGT GTGCCAGCMGCCGCGGTAA 515Fbar23 102115AK515F 11.129S6.WT.Acidified.2041.2.3m 12.129S6.WT.Acidified.2194.2.3m GATCAGCT GTGCCAGCMGCCGCGGTAA 515Fbar24 102115AK515F 12.129S6.WT.Acidified.2194.2.3m 13.129S6.WT.Acidified.2194.3.3m GATCAGGA GTGCCAGCMGCCGCGGTAA 515Fbar25 102115AK515F 13.129S6.WT.Acidified.2194.3.3m 14.129S6.WT.Acidified.2268.2.3m GATCCAAC GTGCCAGCMGCCGCGGTAA 515Fbar26 102115AK515F 14.129S6.WT.Acidified.2268.2.3m 15.129S6.WT.Acidified.2325.3.3m GATCCATG GTGCCAGCMGCCGCGGTAA 515Fbar27 102115AK515F 15.129S6.WT.Acidified.2325.3.3m 16.129S6.WT.Acidified.2041.1.2.6m GATCCTAG GTGCCAGCMGCCGCGGTAA 515Fbar28 102115AK515F 16.129S6.WT.Acidified.2041.1.2.6m 17.129S6.WT.Acidified.2194.2.6m GATCCTTC GTGCCAGCMGCCGCGGTAA 515Fbar29 102115AK515F 17.129S6.WT.Acidified.2194.2.6m 18.129S6.WT.Acidified.2194.3.6m GATCGAAG GTGCCAGCMGCCGCGGTAA 515Fbar30 102115AK515F 18.129S6.WT.Acidified.2194.3.6m 19.129S6.WT.Acidified.2268.2.6m GATCGATC GTGCCAGCMGCCGCGGTAA 515Fbar31 102115AK515F 19.129S6.WT.Acidified.2268.2.6m 20.129S6.WT.Acidified.2325.3.6m GATCGTAC GTGCCAGCMGCCGCGGTAA 515Fbar32 102115AK515F 20.129S6.WT.Acidified.2325.3.6m 21.Cln3KO.Tap.2096.2.3m GATCGTTG GTGCCAGCMGCCGCGGTAA 515Fbar33 102115AK515F 21.Cln3KO.Tap.2096.2.3m 22.Cln3KO.Tap.2100.1.3m GATCTCCT GTGCCAGCMGCCGCGGTAA 515Fbar34 102115AK515F 22.Cln3KO.Tap.2100.1.3m 23.Cln3KO.Tap.2165.2.3m GATCTCGA GTGCCAGCMGCCGCGGTAA 515Fbar35 102115AK515F 23.Cln3KO.Tap.2165.2.3m 24.Cln3KO.Tap.2213.1.3m GATCTGCA GTGCCAGCMGCCGCGGTAA 515Fbar36 102115AK515F 24.Cln3KO.Tap.2213.1.3m 25.Cln3KO.Tap.2213.2.3m GATCTGGT GTGCCAGCMGCCGCGGTAA 515Fbar37 102115AK515F 25.Cln3KO.Tap.2213.2.3m 26.Cln3KO.Tap.2096.2.6m GATGACCT GTGCCAGCMGCCGCGGTAA 515Fbar38 102115AK515F 26.Cln3KO.Tap.2096.2.6m 27.Cln3KO.Tap.2100.1.6m GATGACGA GTGCCAGCMGCCGCGGTAA 515Fbar39 102115AK515F 27.Cln3KO.Tap.2100.1.6m 28.Cln3KO.Tap.2165.2.6m GATGAGCA GTGCCAGCMGCCGCGGTAA 515Fbar40 102115AK515F 28.Cln3KO.Tap.2165.2.6m 29.Cln3KO.Tap.2213.1.6m GATGAGGT GTGCCAGCMGCCGCGGTAA 515Fbar41 102115AK515F 29.Cln3KO.Tap.2213.1.6m 30.Cln3KO.Tap.2213.2.6m GATGCAAG GTGCCAGCMGCCGCGGTAA 515Fbar42 102115AK515F 30.Cln3KO.Tap.2213.2.6m 31.Cln3KO.Acidified.2047.2.3m GATGCATC GTGCCAGCMGCCGCGGTAA 515Fbar43 102115AK515F 31.Cln3KO.Acidified.2047.2.3m 32.Cln3KO.Acidified.2056.5.3m GATGCTAC GTGCCAGCMGCCGCGGTAA 515Fbar44 102115AK515F 32.Cln3KO.Acidified.2056.5.3m 33.Cln3KO.Acidified.2144.2.3m GATGCTTG GTGCCAGCMGCCGCGGTAA 515Fbar45 102115AK515F 33.Cln3KO.Acidified.2144.2.3m 34.Cln3KO.Acidified.2161.2.3m GATGGAAC GTGCCAGCMGCCGCGGTAA 515Fbar46 102115AK515F 34.Cln3KO.Acidified.2161.2.3m 35.Cln3KO.Acidified.2222.2.3m GATGGATG GTGCCAGCMGCCGCGGTAA 515Fbar47 102115AK515F 35.Cln3KO.Acidified.2222.2.3m 36.Cln3KO.Acidified.2047.2.6m GATGGTAG GTGCCAGCMGCCGCGGTAA 515Fbar48 102115AK515F 36.Cln3KO.Acidified.2047.2.6m 37.Cln3KO.Acidified.2056.5.6m GATGGTTC GTGCCAGCMGCCGCGGTAA 515Fbar49 102115AK515F 37.Cln3KO.Acidified.2056.5.6m 38.Cln3KO.Acidified.2144.2.6m GATGTCCA GTGCCAGCMGCCGCGGTAA 515Fbar50 102115AK515F 38.Cln3KO.Acidified.2144.2.6m 39.Cln3KO.Acidified.2161.2.6m GATGTCGT GTGCCAGCMGCCGCGGTAA 515Fbar51 102115AK515F 39.Cln3KO.Acidified.2161.2.6m 40.Cln3KO.Acidified.2222.2.6m CCTAGGCA GTGCCAGCMGCCGCGGTAA 515Fbar52 102115AK515F 40.Cln3KO.Acidified.2222.2.6m