title: GSM3692186: aD18 (ATAC-Seq); Homo sapiens; ATAC-seq
library_construction_protocol: Place frozen tissue into a pre-chilled 2 ml Dounce with 2 ml cold nuclei lysis buffer. Allow frozen tissue to thaw for 5 minutes. Dounce with A pestle until resistance goes away ( 10 strokes). Dounce with B pestle for 20 strokes. Pre-clear larger chunks by pelleting at 100 RCF for 1 min in a pre-chilled centrifuge.Count nuclei using Trypan blue staining and aliquot nuclei for ATAC reaction. Harvest and count cells (counting protocol to be de_ned by the user; see APPENDIX3F). Cells should be intact and in a homogenous, single-cell suspension; Centrifuge 50,000 cells 5 min at 500 x g, 4?C. The number of cells at this step is crucial, as the transposase-to-cell ratio determines the distribution of DNA fragments generated. Remove and discard supernatant. Wash cells once with 50 ?l of cold PBS bu_er. Centrifuge 5 min at 500x g, 4?C. Remove and discard supernatant. Gently pipet up and down to resuspend the cell pellet in 50 ?l of cold lysis bu_er. Centrifuge immediately for 10 min at 500 xg, 44?C. Discard the supernatant, and immediately continue to transposition reaction. Make sure the cell pellet is set on ice. To make the transposition reaction mix, combine the following: TD (2x reaction bu_er from Nextera kit) 25 ?l; TDE1 (Nextera Tn5 Transposase from Nextera kit) 2.5 ?l; Nuclease-free H2O 22.5 ?l. Resuspend nuclei pellet (from step 5) in the transposition reaction mix. Incubate the transposition reaction at 37?C for 30 min. Gentle mixing may increase fragment yield. Immediately following transposition, purify using a Qiagen MinElute PCR Puri_cation Kit. Elute transposed DNA in 10 ?l Elution Bu_er (Bu_er EB from the MinElute kit consisting of 10 mM Tris?Cl, pH 8). To amplify transposed DNA fragments, combine the transposed DNA (10 ?l), nuclease-free H2O (10 ?l),25 ?M PCR Primer 1 (2.5 ?l), 25 ?M Barcoded PCR Primer 2 (2.5 ?l), NEBNext High-Fidelity 2x PCR Master Mix (2 5 ?l). Thermal cycle as follows 72?C,5 min, 1 cycle; 98?C,30 sec; 98?C, 10 sec 5 cycles; 63?C, 30 sec; 2?C, 2 min; 4?C. The library is constructed under the protocol of Iillumina
attributes.tag: GEO Accession
attributes.value: GSM3692186