Fragment analyzer system

The mHVRs for TEDa2cl4 were amplified according to the protocol and primers proposed in [33 (link)]. Amplicons were then purified using the magnetic beads Agencourt AMPure XP-PCR Purification (Beckman Genomics, USA). The concentration of the purified amplicons was controlled using Qubit Fluorometer 2.0 (Invitrogen, USA). The library was validated using the Fragment Analyzer system (Advanced Analytical Technologies, USA). The library was sequenced on an Illumina MiSeq using a 500 cycle v2 kit (Illumina, San Diego, USA). The mHVR reads from TEV55cl1 and PalDa20cl3 were previously published in [33 (link)].

Two biological replicates were performed for each ChIP-seq experiment. ChIP-seq experiments were performed as in ref. ^{20 (link)}. Briefly, nuclei were extracted in lysis buffer (140 mM NaCl, 15 mM HEPES [pH 7.6], 1 mM EDTA, 0.5 mM EGTA, 1% TritonX100, 0.5 mM DTT, 0.1% Sodium Deoxycholate, 10 mM Sodium Butyrate, 1× Protease Inhibitors) and subsequently subjected to ultrasound treatment (Covaris E220, 45 s, peak power 75, duty factor 10, cycles burst 200). Fixed chromatin was then sheared using Covaris E220 (900 s, peak power 140, duty factor 5, cycle burst 200) and precleared overnight. Each sample was incubated with the relevant antibody and concentration (See Supplementary Table 1) for at least 4 h at 4 °C. Samples were then washed, eluted and decrosslinked overnight by incubation at 65 °C. Next, samples were treated with RNaseA (50 μg/mL final concentration) for 30 min at 37 °C and ProteinaseK (200 μg/mL, final) for 3 h at 56 °C. DNA was purified and libraries were prepared according to manufacturer’s instructions using the NEBNext Ultra II DNA Library Prep Kit for Illumina. Libraries were quality controlled by capillary electrophoresis on the Fragment Analyzer system (Advanced Analytical). Sequencing was done using the HiSeq, NextSeq or NovaSeq Illumina platform together with the paired end sequencing option.

RNA extraction was performed using the Quick-RNA^TM MiniPrep kit (Zymo Research, Irvine, CA, USA) and 200 µL of purified viral particles. Reverse transcription and double-stranded synthesis were carried out using the Maxima H Minus Double-Stranded cDNA Synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA) and 13 µL of extracted RNA. Nextera™ DNA Flex Library Preparation kit (Illumina, San Diego, CA, USA) was used from 1 ng of double-strand cDNA. The libraries were purified with AMPure XP (Beckman Coulter, Indianapolis, IN, USA) and quantified with the Qubit dsDNA HS assay kit (Thermo Fisher Scientific, Waltham, MA, USA). The library’s quality and length were assessed with the Agilent high-sensitivity DNA kit (Agilent, Santa Clara, CA, USA) using a Fragment Analyzer™ System (Advanced Analytical Technologies, Inc., Heidelberg, Germany, Europe). Sequencing was performed on the MiniSeq and MiSeq platforms (Illumina, San Diego, CA, USA).

For bulk RNA-seq analysis, total RNA was extracted from FACS-sorted cells using a RNeasy Micro Kit (Qiagen, 74004). Strand-specific libraries were generated using an NEBNext Ultra RNA Library Prep Kit (NEB, E7530) following the manufacturer’s protocol. Libraries of 350±20 bp were obtained, and the quality was determined using a Fragment Analyzer system (Advanced Analytical).
Barcoded libraries were subjected to 150 bp paired-end sequencing on an Illumina HiSeq 2500, and the paired-end reads were aligned to the mouse reference genome (Version mm9 from UCSC) using Tophat (v2.0.13)(Trapnell et al., 2009 (link)). The expression value was generated as the number of fragments per kilobase of transcript per million mapped reads (FPKM) using Cufflinks (v2.2.1) (Trapnell et al., 2012 (link)).

Genomic DNA was extracted from PDX, FFPE, and plasma cell-free DNA using the QIAamp DNA mini kit, QlAamp DNA FFPE Tissue Kit (Qiagen), and QIAamp DNA blood mini kit (Qiagen), respectively, according to the manufacturer’s instructions. Extracted DNA samples were quantified using a NanoDrop or Qubit™ dsDNA HS Assay Kit (Invitrogen). Genomic DNA integrity was determined with the Fragment Analyzer™ system (Advanced Analytical Technologies, Inc).