title: GSM4138615: H3_e161; Homo sapiens; RNA-Seq
library_construction_protocol: Samples were snap-frozen before sonication of chromatin in a Diagenode Bioruptor (4°C, 30s ON / 90s OFF x30 cycles on high) and checked for correct size on an agarose gel. Sonicated samples were pre-cleared before IP with Protein A dynabeads (Invitrogen 100-02D) by rotating 1h at 4°C. Biotinylated chromatin was isolated with T1 streptavedin magnetic beads (Invitrogen 656-01) in the presence of RNase inhibitors (100U/mL)(NEB M0314L) and decross-linked in TE buffer (incl. 0,5% SDS and Proteinase K) 37°C 10h followed by 65°C 6h (interval shaking) for RNA isolation. RNA was purified using a miRNeasy Purification Kit including an on-column DNAse digestion (QIAGEN, Hilden, Germany) as per kit protocol for low RNA amounts. RNA for the long RNA-seq was further purified and concentrated using a Clean and Concentrator-5 kit (Zymo Research, CA, USA) as per kit protocol. Short RNA-seq: 10 ng RNA was used for library preparation with the Diagenode CATS small RNA-seq according to manufacturers protocol, using 10 PCR cycles. Library size selection was performed with AgencourtAMPure XP beads using a 1,4 volume ratio to remove primer-dimers. Libraries were pooled according to experimental replicates with 8 samples at equimolar concentrations including 2% phiX.
attributes.tag: GEO Accession
attributes.value: GSM4138615