title: GSM2056342: sample 51_RNA_GT_ILC2_rpt1; Mus musculus; RNA-Seq
library_construction_protocol: RNA-seq was performed as described previously with slight modification (Hirahara et al., Immunity 2015). Total RNA was prepared from approximately 30-50,000 cells by using TRIzol following manufacture's protocol (Life Technologies). RNA-seq: Total RNA was subsequently processed to mRNA-seq library using TruSeq SR mRNA sample prep kit (FC-122-1001, Illumina). ChIP-seq: Cells were chemically cross linked by 1% formaldehyde. Cell lysates were made by appropriate sonication and protein-DNA complexes were isolated with antibody. ATAC-seq was performed according to published protocol (Buenrostro et al., 2013) with minor modification. 50,000 cells were pelleted and washed with 50ul 1xPBS, followed by treatment with 50ul lysis buffer (10mM Tris-HCl, pH7.4, 10mM NaCl, 3mM MgCl2, 0.1% of IGEPAL CA-630). After pelleting the nuclei by centrifuging at 500x g for 10min, the pellets were re-suspended in 40ul transposition reaction with 2ul Tn5  transposase (Illumina Cat# FC-121-1030) to tag and fragmentalize accessible chromatin. The reaction was incubated at 37ºC with shaking at 300rpm for 30min. The fragmentalized DNAs were then purified using Qiagen MinElute Kit and amplified with 10-11 cycles of PCR based on the amplification curve. Once the libraries are purified using Qiagen PCR Cleanup Kit, they were further sequenced for 50 cycles (paired-end reads) on HiSeq2500. RNA-Seq: The purified libraries were multiplexed and captured on an Illumina flow cell for cluster generation. Libraries were sequenced for 50 single read cycles on HiSeq 2000 or 2500 following the manufacturer's protocols. ChIP-seq: Libraries were prepared according to Illumina's instructions. Briefly, DNA was end-repaired and the blunt-ended, then phosphorylated ends were treated with Taq polymerase and dATP to yield a protruding 3- 'A' base for ligation of NEBNext adapters which have a single 'T' base overhang at the 3' end. After adapter ligation, DNA was PCR amplified with NEBNext index primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel, and quantitated by Qubit (Invitrogen). ChIP-Seq (size fractionation). The purified libraries were multiplexed and captured on an Illumina flow cell for cluster generation. Libraries were sequenced for 50 single read cycles on GAII, HiSeq 2000 or 2500 following the manufacturer's protocols.
attributes.tag: GEO Accession
attributes.value: GSM2056342