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1.
Figure 6

Figure 6. From: Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing.

Cse4p is found robustly at centromeres. All biological replicates were strongly and tightly bound to centromeres, as it is depicted here in the case of CEN11. Two barcoded replicates (Cse4_Rep2, dark blue; Cse4_Rep1, red) and a non-barcoded replicate (Cse4_Rep3, green) were compared to input DNA (light blue). Cse4 ChIP samples were scored against a pool of input DNA. IGB signal tracks of the CEN11 on chromosome 11 are shown for each sample. CEN11 is highlighted in a yellow box. Axis and scale normalizations are similar to Figure 2.

Philippe Lefrançois, et al. BMC Genomics. 2009;10:37-37.
2.
Figure 5

Figure 5. From: Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing.

PolII signal profiles recapitulate findings from Steinmetz et al. PolII ChIP-Seq signal profiles resemble very closely to those published in Figure 3 of Steinmetz et al [54]. We obtained consistent binding at the Bap2-Tat1 loci (a) and at the Sed1-Shu2 loci (b). As expected, we did not observe binding at the Flo11 locus (c). For PolII ChIP-Seq experiments, two biological replicates were barcoded with ACGT (PolII_Rep1, dark blue; PolII_Rep2, orange), one was barcoded with TGCT (PolII_Rep3, red) and a fourth replicate had non-barcoded adapters (PolII_Rep4, green). Input DNA serves as a reference (light blue). Axis and scale normalizations are similar to Figure 2. ORFs above the coordinates axis are on the Watson strand while ORFs below this axis are on the Crick strand.

Philippe Lefrançois, et al. BMC Genomics. 2009;10:37-37.
3.
Figure 2

Figure 2. From: Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing.

Comparison of input DNA signal tracks among all four barcoded adapters relative to standard Illumina adapters. An input sample was split in five aliquots. Four were barcoded differentially (top four lanes) and one had non-barcoded, Illumina adapters (fifth lane, labeled 'None'). Barcoded inputs were scored against non-barcoded input. IGB signal tracks of yeast chromosome 16 are shown for each sample, with ORF locations on the x-axis. ORFs are depicted in purple. On the y-axis, a normalized scale represents the number of read counts at a particular location. Each scale is normalized according to the number of mapped reads (Table 10). A box in the left panel depicts the enlarged section shown in the right panel for positions between 828,000 and 833,000 to demonstrate the overlap among all signal tracks.

Philippe Lefrançois, et al. BMC Genomics. 2009;10:37-37.
4.
Figure 4

Figure 4. From: Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing.

Ste12 distribution during pseudohyphal growth is similar across three different biological replicates. Two barcoded replicates (Ste12_Rep2, dark blue; Ste12_Rep1, red) and a non-barcoded replicate (Ste12_Rep3, green) were compared to input DNA (light blue). Ste12 ChIP samples were scored against a pool of input DNA. IGB signal tracks of chromosome 2 between 340,000 and 410,000 are shown for each sample. Axis and scale normalizations are similar to Figure 2. A box in the left panel containing the TEC1 gene and its surrounding intergenic region was enlarged in panel B and rescaled to emphasize the strong signal at the TEC1 promoter. The same normalization as in Figure 2 was applied. Ste12p and Tec1p act as a dimer during pseudohyphal growth [31].

Philippe Lefrançois, et al. BMC Genomics. 2009;10:37-37.
5.
Figure 7

Figure 7. From: Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing.

qPCR analysis for ChIP samples Cse4-3HA (a), Ste12-13Myc (b) and RNA polymerase PolII (c). For all qPCR analysis, normalization using the 2-ΔΔCp method was used to compare results from a given primer pair to a negative primer pair (respectively Cse4P2, Ste12P1 and Pol2P4) (Table 9). Error bars represent standard deviation across three biological replicates for relative enrichment to the negative primer pair. (a) Cse4p is enriched preferentially at the centromeres (Cse4P1 for CEN3 and Cse4P4 for CEN6) but not at two random non-centromeric locations (Cse4P2 and Cse4P3). (b) Ste12p binds known target sites in pseudohyphal growth. Ste12P1, Ste12P2, Ste12P3 and Ste12P4 represent respectively sites with no enrichment, low enrichment, medium enrichment and high enrichment as determined by ChIP-chip studies (Christopher M. Yellman, unpublished data). While the low enrichment site was not found to be significantly enriched for this ChIP sample, the medium and high enrichment sites were strongly present in our samples used for qPCR. (c) PolII primer pairs were selected using Steinmetz microarray data [54,64] to have three positive pairs (Pol2P1, Pol2P2 and Pol2P3) and one negative pair (Pol2P4). Positive targets were all significantly enriched over the negative control.

Philippe Lefrançois, et al. BMC Genomics. 2009;10:37-37.
6.
Figure 1

Figure 1. From: Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing.

Scheme for yeast barcoded ChIP-Seq. (a) Barcoded ChIP-Seq workflow. Ovals depict yeast cells and squares depict proteins. An aliquot of sheared cell lysate is not immunoprecipitated but is otherwise processed normally (green). This DNA, termed input DNA, is a reference sample for ChIP-Seq. Illumina DNA libraries are generated from both ChIP and input DNA samples. In multiplex ChIP-Seq, a barcoded adapter is ligated to an individual DNA sample. The barcode has 3 unique bases followed by a 'T' to anneal with the end-repaired DNA. Four libraries are then pooled together and applied to a single flowcell lane. After sequencing on the Genome Analyzer, reads are separated according to the first four bases and aligned to the yeast genome. Reads are stacked to generate a signal profile and scored against a pool of input DNA to determine significant transcription factor binding sites. (b) The barcode (orange) is located between Illumina adapter sequences (purple) and ChIP or input DNA inserts (black). The sequencing primer (pink) anneals to the adapter sequences and short reads start with the four bases of the barcode (orange) followed by DNA inserts (black). For the sequencing primer and Illumina adapter, oligonucleotide sequences were given by the manufacturer © 2006 Illumina, Inc. All rights reserved.

Philippe Lefrançois, et al. BMC Genomics. 2009;10:37-37.
7.
Figure 3

Figure 3. From: Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing.

Barcoded adapters perform similarly to standard Illumina adapters and do not crossover to other samples in the same lane. (a) RNA PolII binding profiles from different biological replicates with the same barcode (PolII_Rep1, dark blue; PolII_Rep3, red), with different barcodes (PolII_Rep2, orange) or without barcode (PolII_Rep4, green) strongly overlap. See also Table 3. Input DNA serves as a reference (light blue). IGB signal tracks of chromosome 5 between 130,000 and 320,000 are shown for each library. A box in the left panel depicts the enlarged section shown in the right panel between positions 298,000 and 309,000 to illustrate the overlap among all PolII signal tracks. (b) Binding profiles from four different libraries pooled and sequenced in the same flowcell lane show very little resemblance. Shown here are the binding profiles for Cse4_Rep2 (dark blue), Ste12_Rep2 (red), PolII_Rep2 (green) and Input_ACGT (light blue). IGB signal tracks of chromosome 12 between 80,000 and 210,000 are shown for each sample. For (a) and (b), axis and scale normalizations are similar to Figure 2. (c) Left: Rank-rank comparison of target lists between all pairwise barcoded replicates for Cse4, PolII and Ste12. The horizontal axis shows the fraction of the two lists being compared and the vertical axis shows the fraction of those targets that agree between a given pair of target lists. All comparisons show strong agreement, although the rank lists for Cse4 differ more than PolII or Ste12 for the second half of their length. Right: Rank-rank comparison between barcoded replicates from the same factors (averaged over all pairwise comparisons) compared to rank-rank comparisons for barcoded replicates between different factors: PolII_Rep1 (ACGT) vs. Ste12_Rep1 (TGCT) and Cse4_Rep2 (CATT) vs. Ste12_Rep2 (GTAT).

Philippe Lefrançois, et al. BMC Genomics. 2009;10:37-37.

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