The effect of DNA quality on the sequencing success of cattle

The extraction of amplifiable DNA is a crucial step for genetic analysis of farm animals. The aim of present study was to determine the quality and the quantity of DNA extracted from blood, milk, hair and tissue in cattle. It has been shown that milk and hair samples can be used as a reliable source for obtaining good quality and quantity of DNA ready for the next generation sequencing (NGS). Commercially available kit, with several modifications, was included in the evaluation. Raw milk or milk somatic cell pellet storage under -80 °C or -20 °C has shown to be a reliable procedure for preservation when large number of milk samples were collected at once. The quality (purity) and the quantity of extracted DNA were measured using a nanophotometer. The adequacy of the DNA extract was assessed by estimating average read depth of all captured bases of whole NGS mitogenome sequences. Although DNA extracted from different types of samples was suitable for NGS analysis, hair samples showed the largest variation of DNA quantity, low purity and DNA integrity. Non-invasive sampling methods such as milk and hair collection can be used for dairy cattle while sampling from free-range requires handling, capturing and caution. Milk and hair can be used for obtaining optimal DNA concentrations for NGS analysis

Perturbation experiments indicate effects of CTCF and SMARCA5 on PU.1 expression.

<p><b>A:</b> Western blot of PU.1 in OCI-M2 after CTCF overexpression at 72 hrs. OCI-M2 cells were transfected with CTCF-encoding plasmid (pCTCF) or pBSK+ as negative control plasmid (Ctrl plasmid). AZA treatment (AZA) is indicated on the top of the lines. <b>B:</b> Western blot of PU.1 in OCI-M2 after downregulation of SMARCA5 at 72 hrs. OCI-M2 cells were transfected with siRNA to SMARCA5 and treated or untreated with AZA. Protein lysates were resolved by SDS/PAGE. Membrane with blotted proteins was immunostained with antibody to CTCF or SMARCA5, and PU.1. ß-actin was used as a control of sample loading. Level of down/up-regulation (small table) was determined by densitometry.</p

CTCF/SMARCA5 are recruited to <i>SPI1</i> locus in myeloid cells and upon AZA treatment in AML.

<p><b>A:</b> Sequence conservation of human <i>SPI1</i> locus (VISTA) generated by aligning with murine DNA. Regulatory regions and positions of ChIP amplicons are numbered in respect to human <i>SPI1</i> TSS. <b>B:</b> ChIP of CTCF and SMARCA5 in mixed myeloid cells. <b>C:</b> ChIP of CTCF and <b>D:</b> SMARCA5 in OCI-M2 without (OCI-M2) or with AZA (OCI-M2 AZA) treatment. Y-axis: ChIP enrichment. X-axis: amplicons (distance relative to <i>SPI1</i> TSS). URE, Upstream Regulatory Element of <i>SPI1</i> gene; ENH, enhancer; ELE, element. Error bars: the standard errors (SE). Asterisks: p-values (t-test, 0.05–0.005).</p

Model of epigenetic regulation of <i>SPI1</i> gene by CTCF and SMARCA5.

<p>CTCF binding site (−14.4 kb) becomes occupied by CTCF and SMARCA5 upon AZA-mediated DNA demethylation in AML blasts. Cohesin member’s recruitment partially overlaps with CTCF/SMARCA5 and display spreading over −11.0 kb and URE of <i>SPI1</i>. More diffuse occupancy of both CTCF and SMARCA5 at <i>SPI1</i> gene that was observed in mixed myeloid cells was not achieved in AML blasts, however the AZA treatment partially restored CTCF/SMARCA5 occupancy. Nevertheless, SMARCA5/CTCF is unable to potentiate <i>SPI1</i> derepression in AML blasts and instead, inhibits <i>SPI1</i> transcription possibly through the enhancer-blocking effect at the −14.4 Enhancer.</p

DNA methylation of CTCF binding site in <i>SPI1</i> locus.

<p><b>A:</b> DNA sequence of the CTCF binding site at −14.4 kb Enhancer region within the <i>SPI1</i> locus (CGs are numbered on the top). <b>B:</b> % of DNA unmethylation identified by sequencing of bisulphite-treated DNA isolated from CD34+ cells of AML/MDS patients (N = 3, information in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087448#pone.0087448.s007" target="_blank">Table S1</a>) and control CD34+ cell donors (N = 1) and mixed myeloid cells (N = 1) was performed at the region −14.4 kb of <i>SPI1</i> locus. The primer sequences are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087448#pone.0087448.s008" target="_blank">Table S2</a>. <b>C:</b> % of DNA unmethylation, data in CD34+ cells of MDS patient without AZA therapy (N = 1) and MDS patient treated by AZA (N = 1). <b>D:</b> % of DNA unmethylation in untreated OCI-M2 and AZA-treated OCI-M2. Y-axis: % of unmethylated CpGs; x-axis: number of CpG; error bars indicate standard errors.</p

SMARCA5 cooperates with CTCF on enhancer-blocking activity of <i>H19</i> promoter.

<p><b>A:</b> schematic representation of the reporter constructs pIHLE (without ICR), pIHLME (with mutated CTCF bindng site at the ICR) and pIHLIE (with ICR that can bind CTCF). ICR, insulator of <i>H19</i> gene; H19, promoter of <i>H19</i> gene; luciferase, luciferase gene; Enh, SV40 enhancer; MUT, insulator with mutated CTCF binding sites; kd, protein knock-down. <b>B:</b> Luciferase activity of reporter constructs in HeLa cells. Luciferase acitivity was normalized on Renilla, the pIHLE was set equal to 1. <b>C:</b> HeLa cells depleted for either CTCF or SMARCA5 (using siRNAs) were analyzed for luciferase activity of reporters. Luciferase activity was normalized on non-specific siRNA (Ctrl siRNA) that was set equal to 1, error bars indicate standard errors (SE), t-test (p<0.05 indicated by star).</p

Ctcf and Smarca5 interact in AML cells.

<p><b>A</b> Ctcf occupancy at ICR. ChIP of Ctcf-siRNA-treated MEL cells (Ctcf siRNA, black bars) or non-specific siRNA (Ctrl siRNA, white) at 72 hrs. Y-axis: specific IP DNA fragment enrichment over control IP (standard error, SE). X-axis: ICR amplicons (relative to H19-TSS). Asterisks: p-values (t test, 0.05–0.005). B Smarca5 occupancy at ICR. MEL-shSmarca5 treated 48 hrs with doxycycline (+DOX, black bars) or untreated (-DOX, white). C Ctcf occupancy is decreased at the ICR upon Smarca5 knockdown. Occupancy of Ctcf (lysates from 1B) determined by ChIP. D Smarca5 at ICR upon Ctcf knockdown. Occupancy of Smarca5 (lysates from 1A) by ChIP. E Knock-down of Smarca5 and Ctcf. Protein lysates from samples 1A and 1B were analyzed by Immunoblotting. Migration of Ctcf, Smarca5, and β-actin bands are indicated. Level of downregulation (bellow blots) was determined by densitometry. F Co-IP of Smarca5 and Ctcf in MEL cells. Antibodies for IP and detection are indicated; asterisk indicates nonspecific signal.</p

Smarca5 regulates Ctcf target genes.

<p><b>A:</b> H19 and Igf2 mRNA expression upon Ctcf knockdown (Ctcf siRNA) or Ctrl siRNA. <b>B:</b> Smarca5 knockdown (+DOX) compared to untreatment (-DOX). RT-PCR analyses (A&B) were done at 72hrs. Y-axis: specific mRNAs relative to Hprt1 levels. <b>C:</b> mRNA levels of PU.1 and Cebpa at 96 hrs (4 days) upon Smarca5 knockdown. Y-axis: specific mRNA relative to average of Hprt1 and Gapdh was normalized on negative control (non-specific siRNA). Error bars: the standard errors (SE). Asterisks: p-values (t-test, 0.05–0.005). D: PU.1 and β-actin expression determined by Immunoblotting at 144 hrs (6 days) upon Smarca5 knockdown. Level of downregulation (bellow blots) was determined by densitometry.</p