Nebnext high fidelity master mix

Library preparations were done according to the NEBNext ®-ChIP-Seq library prep reagent set for Illumina protocol (New England Biolabs, Ipswich, USA). For multiplex sample preparation the NEBNext® Multiplex oligos (primer set 1 and 2) (New England Biolabs, Ipswich, USA) were used and libraries were PCR amplified by the NEBNext®High Fidelity Master Mix (New England Biolabs, Ipswich, USA). Libraries were pooled in equimolar ratios and sequenced using the Illumina HiSeq 2000 platform (read length = 50 b) (Illumina, SanDiego, USA). For further analysis only the demultiplexed and quality filtered reads (Eland, Illumina, SanDiego, USA) were used.

The sgRNA inserts were obtained by two rounds of polymerase chain reaction (PCR) amplification from gDNA. The first round was performed with primer pairs listed in Supplementary Table S2 with 16 cycles using NEBNext High Fidelity Master Mix (NEB, # M0541) for the screens with EpiC and Tiling libraries and NEBNext Ultra II Q5 Master Mix (NEB, #M0544) for the screens with the T1 library. A 40 μg aliquot of gDNA was used as the template in eight PCRs to achieve ∼500-fold coverage. The resulting products were purified with a PCR purification kit (Qiagen) and 5 μl of purified products were used as the template for the second round of PCR with 12 cycles. The PCR products were gel purified, quantified by Qubit and qPCR, and deep sequenced using Nextseq500 at MDACC-Smithville Next Generation Sequencing Core. Single-end (75 bp) sequencing was conducted for EpiC and Tiling libraries, and paired-end (75 bp) sequencing was used for the T1 library.

Equal numbers of THP-1 monocytes and THP-1-derived macrophages were collected (50,000 cells per ATAC-seq experiment) and washed with 1× ice-cold PBS. Cells were pelleted via centrifugation (500 × g, 5 min, 4 °C) and resuspended in cell lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 0.1% IGEPAL CA-630), and immediately spun down (500 × g, 10 min, 4 °C). The supernatant was then discarded, and transposition reaction carried out for 30 min at 37 °C with Tn5 transposase in transposition buffer (Illumina, cat#FC-121-1030). DNA was immediately purified on a minElute column (Qiagen), followed by PCR amplification using the NEBNext high-fidelity master mix (NEB cat#M0541) with nextera PCR primers and barcodes. PCR amplification was monitored as described [58 (link)], and gel purified to remove contaminating primer-dimer species.

Mammary gland cells were isolated as described in Additional Methods, section CITE-Seq. 0.5 ug of each Total Seq antibodies (anti-EpCAM and anti-CD49f, Additional file 6: Table S5) was added to the cell suspension and the samples were incubated 30 min at 4 °C. Cells were washed in PBS + BSA 2% + Tween 20 0,01%, counted and adjusted to a concentration of 1000 cells/ µL. Cells were subjected to the Chromium single cell 3’ assay v3 (10X Genomics) as recommended by the manufacturer. After cDNA amplification, ADT-derived cDNAs and mRNA-derived cDNAs were separated based on their size using 0.6 × AMPure XP Beads (Beckman Coulter). The mRNA-derived cDNAs contained in the bead fraction were further processed following the standard 10X Genomics protocol in order to generate single-cell (sc)RNA libraries. The ADT-derived cDNAs contained in the supernatant were further purified (see Additional Methods) and used as template in a PCR reaction with the NEBNext® High Fidelity Master Mix (NEB), a Truseq small RNA RPIx (containing i7 index) primer and the 10X Genomics SI-PCR primer (see Additional file 6: Table S6) to generate the ADT sequencing library. The scRNA-Seq libraries and the ADT libraries were then sequenced together on an Illumina NextSeq500 platform (details Additional file 9 in additional methods).

Genomic DNA was extracted from gastric biopsy samples using MasterPure^TM DNA purification kit (Epicentre, Madison, WI, USA) according to manufacturer’s instructions. The V3–V4 region of 16S rRNA gene was amplified using S-D-Bact-0341-b-S-17 and S-D-Bact-0785-a-A-21 primers designed to include the Illumina-compatible adaptors [17 (link),18 (link)]. The 16S amplicon libraries were prepared according to Illumina 16S library preparation protocol [19 ]. Initial PCR amplification was performed using NEBNext High-Fidelity Master Mix (New England Biolabs, Ipswich, MA, USA) with the following conditions: An initial denaturation at 98 °C for 30 s, followed by 30 cycles consisting of denaturation (98 °C for 10 s), annealing (60 °C for 2 min) and extension (72 °C for 20 s), and a final extension step at 72 °C for 1 min. Automated cluster generation and a 2 × 250 bp paired-end sequencing (MiSeq 500-cycle reagent kit V2) was carried out on the MiSeq platform (Illumina, San Diego, CA, USA) at the Monash University Malaysia Genomics Facility. Metagenomics data reported in this paper are available for public access through the MG-RAST server. The accession number and demographic characteristics of each sample are listed in Table S1.