title: GSM4017304: FLAG IP_b; Cryptococcus neoformans var. neoformans XL280; ChIP-Seq
library_construction_protocol: RNA-Seq: Frozen pellets were ground in liquid nitrogen and total RNA was purified using the Ultrapure RNA Kit (Kangweishiji, CW0581S) according to the manufacturer's instructions, and then reverse-transcribed with the Fastquant RT Kit (Tiangen KR106-02, with gDNase). RNA-Seq: Illumina cDNA libraries were generated using VAHTSTM mRNA-seq v2 Library Prep Kit (Vazyme Biotech Co., Ltd, Nanjing, China) as described by the manufacture. Samples were clustered using VAHRS RNA adapters set1/set2 and then sequenced using Illunima Hiseq 4000 platform in a 2x150 pair-ended manner. ChIP-Seq: Cell lysates were resuspended in MNase reaction Buffer (50 mM Tris-HCl (pH 7.9), 5mM CaCl2), and the protein concentration of each lysate was quantified using BCA assay (#) described as manufacturer's instructions. For each sample, the protein concentrations was diluted to 1 mg ml-1 with total volume of 4 microliters. Chromatin was digested in 2 U μl-1 MNase (#) for 15 min at 37 °C, followed by quenching with 50 μl 0.5 M EDTA. Pellet nuclei and resuspend nuclei in 2 microliters lysis buffer ChIP-Seq: For library construction, 5 ng of raw ChIP material was used for library construction. Illumina libraries were constructed using a modified custom paired-end protocol28. In brief, samples were end-repaired (1_ T4 DNA ligase buffer, 0.4mM dNTP mix, 2.25U T4 DNA polymerase, 0.75U Klenow DNA polymerase and 7.5U T4 polynucleotide kinase; 30 min at 21–25 _C), A-tailed (1_ NEB buffer 2, 0.4mM dNTPs and 3.75U of Klenow (exo-); 30 min at 37 _C) and ligated (1_ rapid DNA ligation buffer, 1mM Illumina PE adapters and 1,600U DNA ligase; 1–8 h at 21–25 _C). Ligated fragments were amplified using indexed primers (Illumina) for 8–10 PCR cycles. DNA was purified with 1.8_volume Ampure XP DNA purification beads between each step
attributes.tag: GEO Accession
attributes.value: GSM4017304