GSM8054460: F123 GAM library f123_1NP_GAM171221_E09_1_S69_R1_001; Mus musculus; OTHER
Fixation of F123 mESC was performed as described previously (Branco and Pombo, 2006). Briefly, cells were grown to 70 % confluency, media was removed, and cells were fixed in 4% and 8% paraformaldehyde in 250 mM HEPES-NaOH (pH 7.6; 10 min and 2 h, respectively), gently scrapped, and softly pelleted and embedded in saturated 2.1 M sucrose in PBS and frozen in liquid nitrogen on copper sample holders. Ultrathin cryosections were cut with a glass knife using an ultracryomicrotome (Leica Biosystems, EM UC7) at ~230 nm thickness, and transferred to PEN membrane steel frame slides 4.0 µm (Leica Microsystems, 11600289) for laser microdissection. Before laser microdissection, cryosections were washed in sterile-filtered 1x PBS (3 times, 5 min each), sterile-filtered H2O (3 times, 5 min each), and stained with sterile-filtered 1 % (w/v) cresyl violet (Sigma-Aldrich, C5042) in H2O for 10 min, followed by 2 washes with H2O (30 s each).  Individual NPs were isolated from the cryosection by laser microdissection using a laser microdissection microscope (Leica Microsystems, LMD7000). Cut NPs were collected in a PCR Cap Strip filled with opaque adhesive material (Carl Zeiss Microscopy, 415190-9161-000). For each collection day, 1 or 2 caps were left empty and taken through the whole genome amplification (WGA) and sequencing process as a negative control for quality control purposes. Whole Genome Amplification (WGA) was performed as described previously (Winick-Ng et al, 2021) with minor modifications. Briefly, DNA was extracted from NPs at 60oC in the lysis buffer (20mM Tris-HCl pH 8.0, 1.4 mM EDTA, 560 mM guanidinium-HCl, 3.5% Tween-20, 0.35% Triton X-100) containing 0.75 units/ml Qiagen protease (Qiagen, 19155). After 24h of DNA extraction, the protease was heat inactivated at 75oC for 30 min and the extracted DNA was amplified via two rounds of PCR. At-first quasi-linear amplification was performed with random hexamer GAT-7N  primers with an adaptor sequence. The lysis buffer containing the extracted genomic DNA was mixed with 2x DeepVent mix buffer (2x Thermo polymerase buffer (10x), 400 µm dNTPs, 4 mM MgSO4 in ultrapure DNase free water), 0.5 µM GAT-7N primers (5′- GTG AGT GAT GGT TGA GGT AGT GTG GAG NNN NNN N) and 2 units/µl DeepVent® (exo-) DNA polymerase (New England Biolabs, M0259L) and incubated for 11 cycles in the BioRad thermocycler. The second exponential PCR amplification was performed in presence of 1x DeepVent mix, 10 mM dNTPs, 0.4 µM GAM-COM primers (5′-GTG AGT GAT GGT TGA GGT AGT GTG GAG) and 2 units/µl DeepVent (exo-) DNA polymerase in the programmable thermal cycler for 26 cycles. After whole genome amplification, the DNA WGA product was purified withSPRI beads (1.7x) The DNA concentration of each sample was quantified using the Quant-iT® PicoGreen dsDNA assay kit (Invitrogen #P7589). Genomic sequencing library was prepared from 1ng of purified DNA using the Illumina Nextera XT library preparation kit (Illumina #FC-131-1096), following the manufacturer's instructions or with reduce to 20% volume of reagents. After the library preparation, DNA was again purified with in-house SPRI beads (1.7x) and equal amounts of DNA from each sample was pooled together (up to 196 samples) for the sequencing. The final pool of libraries was additionally purified two more times with SPRI beads (1.7x) and analyzed using DNA High Sensitivity on-chip electrophoresis on an Agilent 2100 Bioanalyzer. The samples were sequenced on Illumina NextSeq500 machine as single-end 75 bp reads, according to manufacturer's instructions.