Targeted Amplicon Bisulphite Sequencing of LINE-1 elements in DPPA3 KO_UHRF1 GFP mESCs and its rescue mutants with doxycycline inducible overexpression system.. LINE-1_TaBAseq DPPA3 mutant library

CRISPR/ Cas9 generated DPPA3 KO endogenously tagged UHRF1-GFP mouse embryonic stem cells (mESCs) are rescued with wild-type and mutant variants of DPPA3_mScarlet protein by doxycycline-inducible overexpression system. Bisulfite sequencing of the LINE-1 targeted region via Illumina sequencing is performed to determine the methylation levels in each cell line. There were isolated 1 million cells for genomic isolation with QIAamp DNA Mini Kit (Qiagen). 500 ng of gDNA was used for bisulfite conversion followed by the instructions of EZ DNA Methylation-Gold Kit (Zymo), which was eluted in a 2x 20 µl Elution Buffer. TaBAseq is based on two sequential PCRs. The first one amplifies locus-specific LINE-1 elements and the second one indexes the sample-specific amplicon with Ilumina’s Truseq and Nextera compatible overhangs. The amplicon-specific PCR was performed in a total volume of 25 µl containing 0.4 µM of forward and reverse primers, 2mM Betaiinitialne (Sigma-Aldrich), 10mM Tetramethylammonium chloride solution (Sigma-Aldrich), 1x MyTaq Reaction Buffer, 0.5 units of MyTaq HS polymerase (Bioline) and 1 µl of bisulfite converted DNA (12.5 ng). The cycling parameters were as follows: 5 min 95 °C for initial denaturation, 40 cycles (95 °C for 20 s, 58°C for 30 s, 72 °C for 25 s), and with the final elongation 72 °C for 3 min. To check for the quality and yield of the PCR reaction, it was run in an agarose gel of 2 %, and to purify the PCR amplicons, it was used CleanPCR beads with 1.8 x the volume of the remaining PCR reaction. The magnetic beads were immobilized with DynaMag-96 Side Magnet (Thermo Fisher) for 5 min. After the supernatant is removed, the beads are washed 2x with 150 µl of fresh 70 % ethanol. After leaving the beads to air-dry for 5 min, DNA was eluted in 15 µl of elution buffer (10 mM Tris-HCL pH = 8.0). DNA concentration was determined using Nanodrop. Indexing the amplicons, another PCR was performed also in a 25 µl total volume, containing 0.08 µM ( 1 µl of a 2 µM stock) of the Indexing Primers, respectively i5 and i7, 1x MyTaq Reaction Buffer, 0.5 units of MyTaq HS Polymerase (Bioline) and 1 µl of the purified PCR purified amplicon. The cycling parameters are as follows: 5 min 95 °C for initial denaturation, 18 cycles (95 °C for 20 s, 55 °C for 30 s, 72 °C for 40 s), and with the final elongation 72 °C for 5 min. Again 2% agarose gel was used to determine the yield and purity of the PCRs. The PCR-indexed amplicons were purified as described above using the CleanPCR magnetic beads. The DNA concentration for each sample was determined with Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher). The final library was created by pooling together in an equimolar ratio all the PCR products in a final concentration of 1 ng / µl of the total library. The final library concentration was once again estimated via Quant-iT PicoGreen, while the size distribution and quality of the library were assessed with Bioanalyzer. Ilumina Miseq was used to sequence the dual-indexed TaBAseq library with a 2 x 300 bp paired-end, with 5% sequencing coverage.