title: GSM3612263: Ecoli 1_RNAseq; Homo sapiens; RNA-Seq
library_construction_protocol: Neutrophil were washed twice with PBS and lysed in Qiagen buffer RLT (from Qiagen kit 74106) + 10uL/mL beta-mercaptoethanol and run through a QiaShredder (Qiagen 79654) Total RNA was purified via RNeasy mini kit (Qiagen 74106) according to the manufactures instructions, including the RNase-free DNase (Qiagen 79254) treatment step. RNA was eluted in H2O, Turbo DNase kit buffer (ThermoFisher/Ambion AM1907) was added to a 1x concentration and RNA was treated with 4 units of Turbo DNase at 37degrees for 30 minutes. Turbo DNase was then treated with inactivation reagent per manufacturer's specifications. mRNA was purified from total RNA using a Dynabead mRNA purification kit (Life Technologies 61006). First strand synthesis was performed using SuperScript III first strand synthesis system (ThermoFisher 18080051) as follows: 100-500ng RNA; 0.5uL Oligo(dT) primer; 0.8uL random hexamer; 1uL 10mM dNTP, H2O to 9.5uL. Mixture was incubated to 70 degrees for 10 minutes then snap frozen. First strand synthesis mix composed of 2uL 10x RT buffer, 4uL 25mM MgCl2, 2uL 0.1M DTT, 0.uL of 120ng/uL ActinomycinD, 40U RNaseOUT, 200U SuperScriptIII was added to the mixture which was then incubated at 25 degrees for 10 minutes, 42 degrees for 45 minutes, 50 degrees for 25 minutes, and 75 degrees for 15 minutes. Unincorporated nucleotides were removed from the mixture using a ProbeQuant G-50 column (Sigma GE28-9034-08). First strand synthesis reaction as then brought to 51uL with H2O and cooled on ice. 24uL of second strand mixture composed of 1uL 10x RT buffer, 2uL 25mM MgCl2, 1uL 0.1M DTT, 2uL of 10mM dATP, dGTP, dCTP, dUTP mix, 15uL of 5x second strand synthesis buffer (New England Biolabs B6117S), 0.5uL E. coli ligase (NEB M0205S), 2uL DNA polymerase I (NEB M0209S), and 0.5uL RNaseH was added and mixture was incubated at 16 degrees for 2 hours. DNA was purified using a DNA clean and concentrator kit (Zymo D4013), and sonicated on a Covaris E220 with the following settings; Duty cycle 10%; Intensity 5; Cycle per burst 200; Time (seconds) 180. Sonicated DNA was purified using a DNA clean and concentrator kit. DNA was prepared for high throughput sequencing using the methodology described above for ChIP-seq, with the addition of 1uL UNG (ThermoFisher/Applied Biosystems N8080096) during USER enzyme treatment.
attributes.tag: GEO Accession
attributes.value: GSM3612263