title: GSM2695726: THP-1 BCG 6h whole; Homo sapiens; RNA-Seq
library_construction_protocol: For pathogens and the second biological replicates of THP-1, 1.5μg of total RNA was used for RNA-seq library preparation. rRNA was removed from total RNA using Ribo-Zero rRNA Removal Kit. Ribo-Zero RNAs were iron fragmented at 85℃. First-strand cDNA was synthesized with random primers harboring adaptor sequence, the randomized hexamer and Terminal-Tagging oligo cDNA using ScriptSeq™ v2 RNA-Seq Library Preparation Kit (epicentre). The cDNAs were then purified and amplified. PCR products corresponding to 150-350 bps were purified by 12% PAGE gel, quantified and stored at -80℃ until used for sequencing. For high-throughput sequencing, the libraries were prepared following the manufacturer's instructions and BCG were first sequenced with illumina GAIIx system for 33 nt single-end sequencing by ABlife. Inc (Wuhan, China). MTB were applied to Hiseq 2000 system for 101 nt pair-end sequencing by Majorbio. Inc (Shanghai, China). The second biological replicates of THP-1 were applied to illumina NextSeq 500 system for 151 nt pair-end sequencing by ABlife. Inc (Wuhan, China).For the first biological replicates of THP-1, total RNA was treated with RQ1 DNase (promega) to remove DNA. The quality and quantity of the purified RNA were determined by measuring the absorbance at 260nm/280nm (A260/A280) using smartspec plus (BioRad). RNA integrity was further verified by 1.5% Agarose gel electrophoresis. For each sample, 5μg of total RNA was used for RNA-seq library preparation. Polyadenylated mRNAs were purified and concentrated with oligo(dT)-conjugated magnetic beads (invitrogen) before used for directional RNA-seq library preparation. Purified mRNAs were iron fragmented at 95℃ followed by end repair and 5' adaptor ligation. Then reverse transcription was performed with RT primer harboring 3' adaptor sequence and randomized hexamer. The cDNAs were purified and amplified and PCR products corresponding to 200-500 bps were purified, quantified and stored at -80℃ until used for sequencing. For high-throughput sequencing, the first biological replicates of THP-1 were prepared following the manufacturer's instructions and applied to illumina NextSeq 500 system for 151 nt pair-end sequencing by ABlife. Inc (Wuhan, China). RNA libraries were prepared for sequencing using standard Illumina protocols
attributes.tag: GEO Accession
attributes.value: GSM2695726