Single-cell ATAC sequencing
Klaus H. Kaestner Lab, Suzanne Shapira
Abstract
Interrogating cell-type specific chromatin accessibility can reveal cis-regulatory elements linked to downstream gene expression patterns. Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-Seq) is a technique that assays genome-wide chromatin accessibility and can reveal nucleosome positioning patterns, map enhancer and promoter regions, or reveal transcription factor binding sites. Single-cell Assay for Transposase-Accessible Chromatin using Sequencing (scATAC-seq) adapts the bulk ATAC-sequencing protocol to separate individual nuclei and assay chromatin state with single-cell resolution. This technique allows for resolution of gene accessibility patterns within specific pancreatic cell types such as α-cells, β-cells, and acinar cells.
Steps
Steps in pre-processing
1. Transfer handpicked islets (approximately 5,000 IEQs) into 15mL conical tube.
2. Add 10mL of 1xPBS w/o Ca2+, Mg2+ (Rockland, MB-008). Centrifuge for 2 min at RT, 180 xg. Aspirate the supernatant.
3. Add 1mL of warm (37°C ) 0.05% Trypsin (Invitrogen, 25300054) to the islets. Pipette up and down with p1000.
4 . Incubate at 37°C for 9 min, or until cells are in single cells. Pipette up and down at t=7 min, 4 min, 2 min, 0 min.
5. Stop the trypsin reaction by adding 1mL of 100% FBS (Hyclone, SH3091003) to the dissociated islets and pass cells through BD FACs tube with strainer top (Corning 352235)
6. Use 1mL of 100% FBS to rinse the tube and pass through the strainer.
7. Transfer cells to 15mL conical. Centrifuge 4 min, 400 xg.
8. Remove the supernatant and wash cells with PBS with 10% FBS. Centrifuge for 4 min, 400 xg.
9. Wash the cells with PBS with 10% FBS and centrifuge for 4 min, 400 xg. Remove the supernatant.
10. Count cells using a countess chamber.
11. For the scATACseq, do the final resuspension in 0.04% BSA in PBS as is consistent with the instructions for using fresh cell in the Nuclei Isolation Protocol.
Links to kits used in post-processing
1. For nuclei isolation the protocol used is: Nuclei Isolation for Single Cell ATAC Sequencing. We target a 5000 nuclei recovery for this protocol.
2. Samples are processed for scATAC seq using Chromium Single Cell ATAC Reagent Kits.
