GSM5059618: Quantseq_ST+Dox_MSX2_iOX_r2; Homo sapiens; RNA-Seq
RNAseq. RNA was extracted using RNeasy mini kit (Qiagen) and treated with DNaseI (Qiagen). Indexed libraries were prepared with 500 ng RNA using QuantSEQ 3' mRNA-Seq Library Prep Kit FWD for Illumina (015.96, Lexogen) according to manufacturer's recommendations. The libraries were pooled and sequenced with a 100-base-pair single-end (MSX2_iOX) and 50-base-pair single-end protocol on Illumina HiSeq 2500 sequencer. ChIPseq. Cells (1-2x108) were fixed in 2mM Di(N-succinimidyl) glutarate (DSG) (80424, Sigma) in PBS at room temperature (RT) for 45 min. After washing in PBS, cells were fixed again in 1% formaldehyde (28908, Sigma) in TS base media at RT for 12 min. Fixation was stopped by adding glycine to a final concentration of 0.125 M. Cells were washed twice in PBS and resuspended in wash buffer 1 (10mM Hepes pH7.5, 10mM EDTA, 0.5mM EGTA and 0.75% Triton X-100) and incubated at 4C for 10min. After pelleting, cells were resuspended in wash buffer 2 (10mM Hepes pH7.5, 200mM NaCl, 1mM EDTA and 0.5mM EGTA) and incubated at 4C for 10min. After pelleting, cells were lysed in the lysis/sonication buffer (150mM NaCl, 25mM Tris pH 7.5, 5mM EDTA, 0.1%Triton, 1% SDS and 0.5% sodium deoxycholate) with complete protease inhibitors (11873580001, Sigma) on ice for 30min. Chromatin was sonicated 15s on /30s off for 23-25 cycles using the UW2070 sonicator (Bandelin) to the average 300-bp fragments. Chromatin was diluted 1/10 with the dilution buffer (150mM NaCl, 25mM Tris pH 7.5, 5mM EDTA, 1% Triton X-100, 0.1% SDS and 0.5 % sodium deoxycholate) containing complete protease inhibitors. Protein G magnetic Dynabeads (10004D, Invitrogen) were blocked with 1mg/ml BSA and tRNA at 4C for 1h and washed with buffer A (150mM NaCl, 25mM Tris pH 8.0, 1mM EDTA, 1% NP-40, 0.1 % SDS and 0.5 % sodium deoxycholate). Chromatin was pre-cleared with pre-blocked beads at 4C for 1h. Four hundred micrograms of chromatin and seven micrograms of antibody (rabbit anti-MSX2 (HPA005652, Sigma) and rabbit normal IgG (NI01, Sigma) were used per each IP. IP was performed overnight at 4C with rotation. Pre-blocked magnetic beads were added next morning for 7-8 h. Beads were washed at 4C with buffer A three times, buffer B (50mM Tris pH 8.0, 500mM NaCl, 0.1% SDS, 0.5 % sodium deoxycholate, 1% NP-40 and 1mM EDTA), buffer C (50mM Tris pH 8.0, 250mM LiCl, 0.5% sodium deoxycholate, 1% NP-40 and 1mM EDTA) and rinsed with TE buffer. DNA was eluted from beads in the elution buffer (1% SDS, 0.1M NaHCO3). Samples were treated with RNAse A and Proteinase K and reverse-crosslinked overnight by incubation for 2h at 37C, followed by 12h at 65C. DNA was purified on the PCR purification columns (Qiagen). Quantseq libraries were generated using the Quantseq 3' FWD kit according to the maunfacturers recommendations. ChIPseq libraries were generated using the NEBnext ultra II DNA library prep kit according to the standard protocol.