title: GSM9055680: LLC1_StealTHY_Tum_1 LLC1 StealTHY IV BL6 TUMOR 1; Mus musculus; OTHER
library_construction_protocol: All tissue samples were processed through a common DNA extraction protocol, as described (Scheidmann et al., 2022). Briefly, tumor and metastasis-carrying organs were lysed in a FastPrep tissue homogenizer (MP) using dedicated matrix beads-containing disposable tubes (MP) in presence of a DNA extraction buffer (50 mM Tris-Cl pH 8.0, 50 mM EDTA, 1% SDS). The lysate was then digested for at least 16 hours at 65 °C in presence of Proteinase K (Qiagen) and then for 30 minutes at 37 °C in presence of RNAase A (Qiagen). Separation of genomic DNA was carried out by first adding pre-chilled 7.5 M ammonium acetate, followed by isopropanol addition to the resulting supernatant, leading the DNA to precipitate. The pellet was then washed with 70% ethanol and the genomic DNA was resuspended in Tris-EDTA buffer (Ambion) and frozen at -20 °C. Finally, DNA extraction from cultivated cells was carried out using the Dneasy kit (Qiagen). For CRISPR genomics applications, DNA samples were quantified using NanoDrop ND-1000 UV-Vis Spectrophotometer (Thermo Fisher). Samples were processed to reach a theoretical coverage of 500–1,000× per target. For mouse samples, 10-15 µg of genomic DNA were amplified in a one-step PCR with barcoded primers targeting the pLentiGuide vector backbone (P5 and P7). The primer sequences in the table below highlight an 8 bp barcode position in blue and vector binding site in bold, mapping between the sgRNA scaffold and the U6 promoter. All pPrimers were ordered as desalted Oligos oligos at IDT (Integrated DNA Technologies)(at Integrated DNA Technologies or at Microsynth) Microsynth and resuspended in nuclease-free water. For human samples, 10 µg of gDNA were pre-amplified in a nested PCR with the external primers targeting the huTHY1insert and the upstream portion of the U6 promoter (primer sequences in table below). In the second PCR step, barcoded primers targeting the pLentiguide vector backbone (P5 and P7) were used as internal primers (primer sequences in table above). For mouse samples, PCR conditions were as follows: 100 µl as reaction volume, KAPA HiFi Hot Start Ready Mix (2X; Roche)0.02 U/µl of Q5 polymerase (New England Biosciences), and 25-31 cycles of amplification. For human samples, only 23-27 cycles of amplification were applied in the first step using KAPA HiFi Hot Start Ready Mix (2X; Roche). Since some samples did not yield any clearly detectable product after the first PCR, all products were purified with AMPure XP Beads (Beckman Coulter) or Sera-Mag Select Beads (2X, Cytiva Europa GmbH) and used as input for a second PCR (23-27 cycles) with the same primers that were used for the first PCR. When loading a second PCR, pre-amplified fragments were re-quantified using NanoDrop. To reach the theoretical coverage after sequencing, 200 ng of pre-amplified product were re-amplified using nested barcoded primers (P5 and P7). PCR conditions for the second PCR were as follows: 200 ng of pre-amplified fragments per 25 µl reaction, 0.02 U/µl of Q5 polymerase (New England Biosciences), and 27 cycles of amplification. The amplified product containing the spacer sequence with a size of 202 bp was purified by SPRI bead cleanup and a right-sided size selection to remove the genomic DNA input. Using a dual barcoding system, the final NGS amplicons were multiplexed and sequenced on an Illumina platform with 21 dark cycles (DC) in Read 1 to skip the invariable region of the primer binding side (U6 promoter).