The increase in miR-100 levels is supported by precise copy number calculations that show that there are 329 molecules of miR-100 per ng of total input RNA in cells grown in the absence of donor cells and those numbers increase to 443 molecules of miR-100 in the presence of donor cells.
Experiments were performed in triplicate for each input (FLAG or H3) and primer pair combination starting from 0.2 ng of input DNA per reaction.
Standard TaqMan assays (20 μL) were performed by using an Applied Biosystems Universal Assay Buffer with 10 ng of input cDNA per reaction and amplified for 40 cycles in an ABI Prism 7900HT Sequence Detection System (SDS software version 2.3).
Genotyping was performed using Illumina 610-quad arrays at the Sanger Institute, UK. 200 ng of input DNA per sample were used and prepared for genotyping using the Illumina Infinium system following the manufacturer's protocols.
Each sample was assayed in quadruplicate with 20 ng of input RNA per well in a 25- μl reaction volume containing 1 × QuantiTect SYBR Green PCR Master Mix and 1 × QuantiTect gene expression assay (Qiagen).
We report a cost-effective and fully scalable, pre-hybridization multiplexed in-solution capture method, amenable to <100 ng of input DNA per person due to removal of sample transfers during library preparation enabled by the constant presence of SPRI beads and resulting in a 50% reduction in the volume of library preparation reagent necessary.
Solution hybridization with MYbaits biotinylated RNA baits (MYcroarray, Ann Arbor, Michigan, USA) and enrichment followed Tennessen et al. (2013) with approximately 350 480 ng of input DNA per pool and 12 rather than 15 cycles of PCR enrichment to decrease the production of PCR duplicates.
Per well, molecularly barcoded, Illumina-compatible specific oligonucleotides containing sequences complementary to the primer tails were added to the Fluidigm Access Array chip together with genomic DNA samples (20 50 ng of input) such that all amplicons for a given DNA sample shared the same index, and PCR was performed according to the manufacturer's instructions.
As mentioned by the authors, "hybridizations from <10 ng of input total RNA had decreased correlation".
In the pS2-methylated cells, MDA MB231 and HeLa, when antibodies against MBD2 were used, the amount of pS2 promoter per ng of total DNA in immunoprecipitated fraction (Figure 2A, "bound") was greater than in the input, or in the non-retained fractions (Figure 2A, "input", "unbound", and "IgG"), indicating that this methylated region is immunoprecipitated by anti-MBD2 antibodies.
