title: GSM2805361: BCL11A_CD34_Day9_30min; Homo sapiens; OTHER
library_construction_protocol: CUT&RUN experiments were carried out as described(Skene and Henikoff, 2017) with minor changes. Briefly, nuclei from 2 million cells were isolated and incubated with primary antibody for 2 hours. After washing away unbound antibody, protein A-MNase was added at a 1:1000 ratio and incubated for 1 hour. The nuclei were washed again and placed in a 0°C metal block. To activate protein A-MNase, CaCl2 was added to a final concentration of 2mM. The reaction was carried out for different time courses and stopped by chelating Ca2+. The protein-DNA complex was released by centrifugation and then digested by proteinase K at 50°C overnight. DNA was extracted by ethanol precipitation, followed by Qubit and bioanalyzer quality control. Protein A-MNase (batch 5) was kindly provided by Dr. Steve Henikoff.  The antibodies used were: GATA1, ab11852, abcam; CTCF, 07-729, Millipore; BCL11A, ab191401, abcam; normal rabbit IgG, 12-370, Millipore. For library preparation, we modified the protocol of NEBNext® Ultra™ II DNA Library Prep Kit, NEB (Ipswich, USA), aiming to preserve small DNA fragments (30-80bp). Briefly, dA-tailing temperature was decreased to 50°C to avoid DNA melting, and the reaction time was increased to 1 hour to compensate for lower enzymatic activity. After adapter ligation, 2.1x volume of AMPure XP beads was added to the reaction to ensure high recovery efficiency of small fragments. After 12 cycle of PCR amplification, the reaction was cleaned up with 1.2x volume of AMPure XP beads. 16-24 barcoded libraries were quantified, mixed and denatured according to the standard protocol from Illumina. 1.3mL of 1.8pM diluted library was loaded to a NextSeq® 500/550 High Output Kit v2 (75 cycles), and sequenced in the NextSeq 500 platform with paired-end sequencing.
attributes.tag: GEO Accession
attributes.value: GSM2805361