GSM2997959: INOF_FRT; Homo sapiens; RNA-Seq
cDNA library produced with TGIRT RNA was isolated and DNase treated using RNEasy mini kit (Qiagen 74106) according to the manufacturer protocol. 5ug DNA-free total RNA was then ribodepleted using Ribo-zero Gold (Illumina RZG1224 ) according to the manufacturer protocol and purified using a modified ZYMO RNA Clean and Concentrator (R1016) protocol where 8 volumes EtOH instead of 4. rRNA depleted RNA was fragmented with NEBNext Magnesium RNA Fragmentation Module (E6150) followed by dephosphorylation using T4PNK (mandel  ) and purified by same modified ZYMO protocol. cDNAs were synthesized via TGIRT template-switching with 1µM TGIRT-III reverse transcriptase (Ingex, LLC) for 15 min at 60o C, during which a DNA oligonucleotide containing the complement of an Illumina Read 2 sequencing primer-binding site becomes seamlessly linked to the 5' cDNA end. After reaction cleanup (Qiagen MinElute Reaction cleanup 28206), a 5' adenylated DNA oligonucleotide containing the complement of an Illumina Read 1 sequencing primer-binding site is then ligated to the 3' cDNA end with Thermostable 5' AppDNA / RNA Ligase (New England Biolabs M0319). Properly ligated cDNAs were amplified by PCR (12 cycles) to synthesize the second strand and add Illumina flowcell capture and index sequences. Library was size-selected with Ampure XP beads (Beckman-Coulter) and quantified with Qubit and evaluated on an Agilent 2100 Bioanalyzer.
GEO Accession
GSM2997959