Omni-ATAC-seq for Chromatin Accessibility Profiling in 50,000 Cryopreserved Human PBMCs Using Tn5 Tagmentation

Three biological donors, two technical replicates (independent tagmentation reactions) per donor, fixed input of 50,000 viable cells per reaction (counted by AOPI viability stain, viability ≥ 80%). Tagmentation time is fixed at 30 min/37°C. A bulk genomic DNA (no-chromatin) tagmentation control and an over-/under-tagmentation titration (25k, 50k, 100k cells) establish the optimal Tn5:cell ratio. Donor identity is barcoded; library prep is randomized across the plate and the operator is blinded to donor during QC.

BSL-2 for human PBMCs (potential bloodborne pathogens — HIV, HBV, HCV). Handle in a Class II biosafety cabinet, wear gloves, lab coat, and eye protection; decontaminate surfaces and pipettes with 10% bleach followed by 70% ethanol. Treat all PBMC waste as biohazardous and autoclave or incinerate. Tn5 and PCR reagents are low hazard; follow standard chemical handling. Avoid bleach contact with guanidinium-containing column buffers.

Positive control: a reference PBMC aliquot with known good TSS enrichment processed in each batch. Negative control: tagmentation of purified genomic DNA (no chromatin structure) should yield uniform, non-periodic fragments and serves as an accessibility-specificity control. No-template PCR control detects index-primer contamination. A digitonin-omitted reaction confirms detergent dependence of permeabilization. Mitochondrial read fraction is an internal QC metric.

TapeStation trace shows clear nucleosomal banding. Post-sequencing: mitochondrial reads <20%, TSS enrichment >7, FRiP >0.3, and >50,000 narrow peaks called per sample. Fragment-size distribution shows characteristic ~10.5 bp helical periodicity and distinct mono-/di-nucleosome peaks. Library yield 5-30 nM. Duplication rate <40% at this input.

• Cryopreserved human PBMCs (Ficoll-isolated), thawed into RPMI-1640 + 10% FBS
• AOPI viability dye + automated cell counter
• Resuspension buffer (RSB): 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2
• Omni lysis buffer: RSB + 0.1% NP-40 + 0.1% Tween-20 + 0.01% digitonin
• Wash buffer: RSB + 0.1% Tween-20
• Loaded Tn5 transposase (Illumina Tagment DNA enzyme TDE1) + 2x TD tagmentation buffer
• PCR: NEBNext/KAPA HiFi 2x master mix + Nextera i5/i7 index primers
• DNA Clean & Concentrate column kit or AMPure XP beads
• 10% Tween-20, EDTA-free protease inhibitor (optional), nuclease-free water
• Qubit dsDNA HS kit; TapeStation HS D1000 reagents

To produce high-quality ATAC-seq libraries from 50,000 viable human PBMCs by Tn5 transposase tagmentation of accessible chromatin, incorporating the Omni-ATAC detergent combination (NP-40 + Tween-20 + digitonin) and an optimized wash to suppress mitochondrial DNA capture below 20%. Libraries should yield clear nucleosomal periodicity (sub-nucleosomal, mono-, di-nucleosome fragments) and a TSS enrichment score > 7.

1. Thaw PBMCs rapidly at 37°C, dilute dropwise into 10 mL warm RPMI+FBS, count with AOPI; require ≥80% viability.
2. Aliquot 50,000 cells; pellet 500 x g, 5 min, 4°C; aspirate.
3. Resuspend in 50 µL cold Omni lysis buffer, pipette gently, incubate on ice 3 min to permeabilize.
4. Add 1 mL cold wash buffer (NO digitonin/NP-40); pellet nuclei 500 x g, 10 min, 4°C; aspirate fully.
5. Resuspend nuclei in 50 µL tagmentation mix (25 µL 2x TD buffer, 2.5 µL TDE1, 16.5 µL PBS, 0.5 µL 1% digitonin, 0.5 µL 10% Tween-20, water).
6. Incubate 37°C, 30 min, 1000 rpm on a thermomixer.
7. Stop and purify with DNA Clean & Concentrate column; elute in 21 µL.
8. Pre-amplify: 5 cycles PCR (72°C 5 min gap-fill; 98°C 30 s; [98°C 10 s, 63°C 30 s, 72°C 1 min] x5).
9. qPCR side-reaction on 5 µL to determine additional cycles (stop at 1/3 max fluorescence; typically +4-6).
10. Complete remaining cycles; purify with 1.1x AMPure XP, elute 20 µL.
11. QC on TapeStation: expect nucleosomal ladder (~50, 200, 400 bp). Quantify by Qubit.

Independent variable: cell state / donor. Dependent variables: TSS enrichment score, fraction of reads in peaks (FRiP), mitochondrial read percentage, nucleosomal periodicity, and number of called peaks. Controlled variables: input cell number (50,000), tagmentation time (30 min) and temperature (37°C), Tn5 lot and amount, detergent concentrations, and PCR cycle number (set by qPCR side-reaction).

If nuclei are gently permeabilized with the Omni-ATAC detergent mix and washed to remove cytoplasmic mitochondria before tagmentation, then the resulting libraries will show reduced mitochondrial reads (<20%) and improved nuclear accessibility signal (TSS enrichment > 7) compared with the original Fast-ATAC, enabling robust peak calling from only 50,000 input cells.

Trim Nextera adapters with fastp/Trim Galore. Align to GRCh38 with Bowtie2 (--very-sensitive -X 2000). Remove duplicates (Picard MarkDuplicates), filter MAPQ<30, remove chrM and ENCODE blacklist regions. Shift reads +4/-5 bp for Tn5. Call peaks with MACS2 (--nomodel --shift -75 --extsize 150 -q 0.01). Compute TSS enrichment and FRiP with ATACseqQC/deepTools. Generate normalized bigWigs (RPKM/CPM) for visualization; perform footprinting with TOBIAS or HINT-ATAC.

1. High mitochondrial reads (>40%): the cytoplasm/mitochondria wash was insufficient — ensure the post-lysis wash with Tween-only buffer is performed and aspirate fully; reduce lysis time. 2. No nucleosomal ladder / smear: over-tagmentation — reduce TDE1 amount or cell number, or shorten incubation. 3. Large fragments only / weak signal: under-tagmentation or low viability — confirm ≥80% viability and increase Tn5. 4. Low FRiP / low TSS enrichment: dead cells or debris — re-thaw with a slower dilution and consider a dead-cell removal step. 5. Adapter dimers in trace: increase AMPure ratio or add a second 1.1x cleanup.

Differential accessibility across conditions with DiffBind/DESeq2 on a consensus peak set, n=3 donors, negative-binomial test with Benjamini-Hochberg FDR at α = 0.05. Use donor as a blocking factor in the design matrix to control for inter-individual variability. Report log2 fold-change and padj. With 3 donors x 2 technical replicates, the design has ~80% power to detect 2-fold accessibility changes at well-covered peaks (mean count >30).