title: GSM2482365: TrpA_PolA; Trypanosoma cruzi; RNA-Seq
library_construction_protocol: Total RNA was extracted from T. cruzi epimastigote and trypomastigote forms (5 × 108 cells) and from confluent HeLa cells with the RNeasy kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions, with an additional on-column DNase digestion step. Total RNA from HeLa cells were obtained from MessageAmpTM II aRNA Amplification Kit from Life Technologies® (#AMB1751-5). RNA integrity was assessed on an Agilent 2100 Bioanalyzer with RNA 6000 Nano LabChip kit, according to the manufacturer’s instructions. Polyadenylated RNA were purified from at least 50 µg of total RNA using PolyA+Track® mRNA Isolation System III from Promega® (#Z5300), according to the manufacturer's instructions. Polyadenylated RNA were purified from at least 50 µg of total RNA using PolyA+Track® mRNA Isolation System III from Promega® (#Z5300), according to the manufacturer's instructions. All mRNA amplification reactions were done using reagents from MessageAmpTM II aRNA Amplification Kit from Life Technologies® (#AMB1751-5). For classic Eberwine amplification method, first-strand cDNA was synthesized using a T7oligo(dT) primer that contains a T7 RNA polymerase promoter upstream to the poly-T tract; this promoter will further direct the in vitro mRNA synthesis (amplification). First, 100 ng of total RNA (in 11 µl) were mixed with first-strand cDNA reaction mix containing 1 µl of T7oligo(dT) primer, 2 µl first-strand buffer, 4 µl dNTPs, 1 µl RNase inhibitor and 1 µl ArrayScript Reverse Transcriptase and incubated for 2 hours at 42ºC. Second-strand cDNA was then synthesized for 2 hours at 16ºC using the total 20 µl first-strand product plus 80 µl of second strand mix containing 63 µl water, 10 µl second-strand buffer, 4 µl dNTPs, 2 µl DNA Polymerase and 1 µl of RNase H. Double-strand cDNA was then purified using PureLink® PCR Micro Kit from Life Technologies (#K310250) according manufacturer’s instructions. Eluted cDNA was adjusted to 16 µl and used as template for amplification reaction in a total volume of 40 µl containing 16 µl NTPs, 4 µl amplification buffer and 4 µl of T7 RNA polymerase; in vitro transcription took 14 hours at 37ºC. Amplified RNA (aRNA) was then purified using RNeasy MinElute CleanUp Kit (Qiagen, #74204), according to the manufacturer’s instructions. For specific amplification of spliced leader containing mRNA, we used a custom designed primer complementary to the last 21 bases of T. cruzi spliced leader (in bold) with an upstream T7 RNA polymerase promoter (in gray) (5'-GGCCAGTGAATTGTAATACG ACTCACTATAGGGAGGCGGTACAGTTTCTGTACTATATTG-3'), which we named T7SL. In this case, T7SL was used for second-strand cDNA synthesis, while first-strand cDNA was produced using random primers, accordingly to the second round amplification protocol of MessageAmpTM II aRNA Kit. Template total RNA was adjusted to 10 µl, mixed with 2 µl of "second round primers" (random primers) and incubated at 70ºC for 10 minutes followed by snap cool on ice to allow annealing of random primers to RNA. Then, 8 µl of reverse transcription master mix was added and first-strand cDNA synthesized at 42ºC for 2 hours. RNase H was added (1 µl) and sample incubated at 37ºC for 30 minutes to specifically degrade the remaining RNA. T7SL primer was added to a final concentration of 1 µM and the sample was incubated at 70ºC for 10 minutes and then placed on ice. Second-strand cDNA master mix without RNase H was added (74 µl) and the reaction incubated for 2 hours at 16ºC. Double-strand cDNA purification, in vitro transcription and aRNA purification was performed as described above. Total RNA-Seq Kit (Life Technologies, #4445374) was used accordingly to manufacturer instructions. Briefly, RNA was fragmented using RNase III and cleaned up with Invitrogen RiboMinus™ Concentration Module. Strand-specific adaptors were hybridized and ligated to both extremities of RNA in an overnight reaction, followed by first-strand cDNA synthesis with reverse transcriptase. cDNA was purified using MinElute® PCR Purification Kit (Qiagen) and gel-based selected for size using a Novex TBE-Urea 6% gel (#EC6865bOX). On-gel cDNA was amplified by PCR using SOLiDTM RNA barcoding kit primers (Life Technologies, #4427046 and #4453189) and purified with Invitrogen PureLink® PCR Micro Kit. Amplified DNA was quantified with Qubit® dsDNA HS Assay Kit on a Qubit® 2.0 Fluorometer. Equal masses of each sample, containing specific barcodes, were pooled together and the mixture used as DNA template for emulsion PCR on Applied Biosystems SOLiD® EZ Bead™ E80 system  (#4453095). After 3' end modification with terminal transferase, template beads were deposited onto glass slides and libraries sequenced on a SOLiDTM 4 system using multiplex fragment sequencing protocol that generates about 700 millions 50 bases short reads per run.
attributes.tag: GEO Accession
attributes.value: GSM2482365