alias: GSM3467412
title: GSM3467412: T.DP.Th.Rorc(γt)GFP/GFP#2; Mus musculus; ATAC-seq
library_construction_protocol: Immunocytes were isolated to high purity by flow cytometry according to ImmGen SOP using the antibodies and gates manifested in ImmGen website (www.immgen.org). After the 2nd rounds of sorting, 10,000 cells (except very rare cell populations including LTHSC.34-.BM, LTHSC.34+.BM and STHSC.150-.BM which were sorted 677, 2483 and 3660 cells respectively) were collected in 100uL of BAMBANKER (serum-free cell freezing medium, No.302-14681, Wako, Osaka, Japan) pre-chilled on ice. Tubes were kept on ice at most 30min until frozen in a cell freezing container with isopropyl alcohol (-1°C/min rate, 4C to -80C) and stored at -80C until preparing ATAC-seq libraries. Frozen cells were thawed, washed with 1mL of PBS containing protease inhibitors (cOmplete EDTA-free protease inhibitor cocktail, Roche Diagnostics, Basel, Switzerland) and cell pellets were resuspended in 10uL of Tn5transposase mixture: 1xTagment DNA Buffer, 0.5uL Tagment DNA Enzyme (Nextera DNA Library Preparation Kit, illumine, CA, USA) and 0.2mg/ml digitonin (#G9441, Promega, WI, USA) on ice. Then, the cells were incubated at 37C for 30min with agitation followed by DNA isolation using MinElute Reaction Cleanup Kit (Qiagen, Hilden, Germany) according to the manufacturer's instruction. To construct libraries, the 1st round of PCR was performed in 50uL using NEBNext High-Fidelity 2X PCR Master Mix (New England Biolabs, MA, USA) with a set of primers (0.5uM each) and following thermal cycles: 5min at 72°C, 30 sec at 98°C, followed by 7 cycles [98°C for 10 sec, 63°C for 30 sec and 72°C for 60 sec] and a final extension at 72°C for 5mn. The PCR products were purified and size-selected using Agencourt AMPure XP beads (Beckman Coulter, CA, USA) (0.65x and 1.8x volume to remove long and short fragments respectively) and eluted in 18uL of EB (Qiagen). For avoiding an over amplification which can produce more GC bias, 2uL of the eluted DNA were subjected to qPCR (StepOnePlus Real-Time PCR System, Life Technologies, CA, USA) in 20uL employing SYBR GreenI (final 0.6x SYBR GreenI, Life Technologies) and the same set of primers (1.25uM each) as in the 1st round of PCR. After running qPCR (30sec at 98°C, followed by 30 cycles [98°C for 10 sec, 63°C for 30 sec and 72°C for 60 sec]), amplification curves were examined and the optimal number of PCR cycles were estimated as the cycles where amplifications reach 1/4 of maximum. 12.5uL of the eluted DNA were subjected to the 2nd round of PCR in 50uL with NEBNext High-fidelity 2x PCR master mix, a set of primers (1.25uM each) and following thermal cycles: 30 sec hot-start at 98°C, followed by 7~13 cycles [98°C for 10 sec, 63°C for 30 sec and 72°C for 60 sec] and a final extension at 72°C for 5mn. The libraries were purified by Agencourt AMPure XP beads (x1.8 vol.), quantified by qPCR employing Power SYBR Green PCR Master mix (ThermoFisher scientific, MA, USA) and a common set of primers (P5_FW and P7_RV, 0.2uM each) and pooled, which were sequenced for paired-end (38+37bp) on an Illumina Next-seq 500 instrument in a high-output mode.
identifiers.namespace: GEO
identifiers.id: GSM3467412