GSM2221734: protective2-4; Homo sapiens; RNA-Seq
RNA extraction with on-column DNase1 digestion was performed using the RNeasy kit (Qiagen) according to manufacturer’s instructions Total RNA (extracted as described above in qRTPCR) was quantified using both, NanoDrop to assess sample contamination by proteins or carryover reagents from RNA isolation, and Qubit fluorometer (Invitrogen). Samples were then qualified using the Fragment Analyzer (Advanced Analytical Technologies, Ankeny, IA) to determine integrity of the total RNA by measuring the ratio between the 18S and 28S ribosomal peaks. RNAseq libraries maintaining strand-specificity were made using 10ng of material per sample with the SMARTer Ultra Low Input RNA v4 kit (Clontech), and indexed using Nextera indices (Illumina), according to the manufacturer’s instructions. Final libraries were again quantitated with the Qubit fluorometer and checked for size via the Fragment Analyzer. Libraries were diluted to 4nM and pooled in equal volumes for denaturation, hybridization, and sequencing on the Illumina NextSeq 500 (Illumina) with paired end 150bp sequencing chemistry .
GEO Accession
GSM2221734