OP-HELISCOPE-CAGE-v4.0

Protocol: OP-HELISCOPE-CAGE-v4.0

Author: Kojima, Miki

Created: Apr. 28, 2011

Update:

Parameters:

Description:

Sample preparation unit is 96 wells.

Sample is prepared by HeliScope CAGE automation system.

Automation machines is TECAN EVO 150 RIKEN customized.

There are 2 machines (EVO NO.1 and EVO NO.2)

<Day1> First-strand cDNA synthesis: First-strand cDNA was synthesized using total RNA, whose RIN number of Bioanalyzer and RNA 6000 Pico kit (Agilent) is greater than 7, and whose concentration is more 0.5 µg/µl. Each aliquot of 5 µg of total RNA was mixed with 100 pmol of random N15 primer and added RNase free water up to 6 µl in well of 96 plate respectively. The RT enzyme mixture contained 1x SuperScript III reaction buffer, 1380 nmol dNTP, 0.65 M sorbitol, 0.14 M trehalose solution, 0.05 mM DTT, 104880 units of SuperScript III (Invitrogen) for a total volume of 4416 µl for 96 reactions and divided this to 180 µl/well in 8 strip tube. AMPure RNA Clean XP beads was prepared 900µl in 8 of 1.5 ml tubes. 100% Ethanol was prepared 30 ml in Falcon tube. 70% Ethanol was prepared 50 ml in falcon tube. H2O(Gibco) was prepared 25 ml in Falcon tube. 96 well plate with sample, new 96 well plate, RT enzyme mixture, AMPure RNA Clean XP beads, 100% Ethanol, 70% Ethanol and H2O(Gibco) were set in TECAN EVO150 Labware stage. The program of “1_1st_STRAND_SYNTHESIS_and_PURIFICATION” was run. The purified 96 samples were collected to new 96 well plate.

<Day2> Oxidation and biotinylation: The Oxidation mixture contained 0.5 M sodium acetate (pH 4.5) and 125 mM sodium periodate for a total volume of 820 µl for 96 reactions and divided to 100 µl/well in 8 strip tube. The Oxidation stop mixture contained 5% Glycerol and 0.875 M Tris HCl (pH 8.5) for a total volume of 10 ml for 96 reactions in 15ml Falcon tube. AMPure RNA Clean XP beads was prepared 1800 µl in 8 of 2.0 ml tubes. Isopropanol was prepared 20 ml in Falcon tube. 100% Ethanol was prepared 30 ml in Falcon tube. 70% Ethanol was prepared 50 ml in falcon tube. H2O(Gibco) was prepared 25 ml in Falcon tube. 96 well plate with sample, new 96 well plate, Oxidation mixture, Oxidation stop mixture, AMPure RNA Clean XP beads, Isopropanol, 100% Ethanol, 70% Ethanol and H2O(Gibco) were set in TECAN EVO150 Labware stage. The program of “2_OXIDATION_and_PURIFICATION” was run. The purified 96 samples were collected to new 96 well plate.

<Day3> Biotinylation: The Biotinylation mixture contained 5 mM Biotin (Long arm) Hydrazaide and 0.5 M sodium acetate (pH 6.0) for a total volume of 1400 µl for 96 reactions and divided to 170 µl/well in 8 strip tube. AMPure RNA Clean XP beads was prepared 1400 µl in 8 of 1.5 ml tubes. Isopropanol was prepared 20 ml in Falcon tube. 100% Ethanol was prepared 30 ml in Falcon tube. 70% Ethanol was prepared 50 ml in falcon tube. H2O(Gibco) was prepared 25 ml in Falcon tube. 96 well plate with sample, new 96 well plate, Biotinylation mixture, AMPure RNA Clean XP beads, Isopropanol, 100% Ethanol, 70% Ethanol and H2O(Gibco) were set in TECAN EVO150 Labware stage. The program of “3_BIOTINILATION_and_PURIFICATION” was run. The purified 96 samples were collected to new 96 well plate.

<Day4> RNase ONE : The RNase ONE mixture contained 1x RNase ONE buffer and 900 unit of RNase ONE for a total volume of 900 µl for 96 reactions and divided to 110 µl/well in 8 strip tube. 96 plate with sample and RNase ONE mixture were set in TECAN EVO150 Labware stage. The program of “4_1_RNaseONE_CAPTRAPPING_RELEASE” was run.

<Day5> Capture and Release: 5600 µl of MPG streptavidin magnetic beads solution (TAKARA Bio) was mixed with 70 µl of 20 µg/µl tRNA (Sigma), then stand on ice for 30 min with occasional mixing to block the bead surfaces. The beads were separated using a magnetic tube stand, then twice washed with 6000 µl of wash buffer 1 containing 4.5 M sodium chloride and 50 mM EDTA (pH 8.0), followed by suspension in 11760 µl of the same buffer with 1400 µg of tRNA included. Washed MPG beads with tRNA was divided to 1450 µl in 8 of 2.0 ml tubes. Wash buffer 2 containing 0.3 M sodium chloride and 1 mM EDTA (pH 8.0) was prepared 50 ml in Falcon tube. Wash buffer 3 containing 1 mM EDTA, 0.4% sodium dodecylsulfate, 0.5 M sodium acetate, and 20 mM Tris-HCl (pH 8.5) was prepared 25 ml in Falcon tube. Wash buffer 4 containing 1 mM EDTA, 0.5 mM sodium acetate, and 10 mM Tris-HCl (pH 8.5) was prepared 50 ml in Falcon tube. 1x RNase ONE buffer for as Release buffer was prepared 50 ml in Falcon tube. 96 plate with sample, MPG beads, Wash buffer 2, Wash buffer 3, Wash buffer 4, 1xRNase buffer were set in TECAN EVO150 Labware stage. The program of “4_2_RNaseONE_CAPTRAPPING_RELEASE” was run. The released cDNA 96 samples were collected to new 96 well plate.

<Day6> RNase H/ONE  : The RNase H/ONE mixture contained 1x RNase ONE buffer, 1080 unit of RNase H, 3600 unit of RNase ONE for a total volume of 900 µl for 96 reactions and divided to 110 µl/well in 8 strip tube. AMPure XP beads was prepared 1650 µl in 8 of 2.0 ml tubes. 100% Ethanol was prepared 30 ml in Falcon tube. 70% Ethanol was prepared 50 ml in falcon tube. H2O(Gibco) was prepared 25 ml in Falcon tube. 96 well plate with sample, new 96 well plate, RNase H/ONE mixture, AMPure XP beads, 100% Ethanol, 70% Ethanol and H2O(Gibco) were set in TECAN EVO150 Labware stage. The program of “5_RNaseH_and PURIFICATION”was run. The purified 96 samples were collected to new 96 well plate.

<Day7> RNase ONE  : The RNase ONE mixture contained 1x RNase ONE buffer and 900 unit of RNase ONE for a total volume of 900 µl for 96 reactions and divided to 110 µl/well in 8 strip tube. AMPure XP beads was prepared 1100 µl in 8 of 1.5 ml tubes. 100% Ethanol was prepared 30 ml in Falcon tube. 70% Ethanol was prepared 50 ml in falcon tube. H2O(Gibco) was prepared 25 ml in Falcon tube. 96 well plate with sample, new 96 well plate, RNase ONE mixture, AMPure XP beads, 100% Ethanol, 70% Ethanol and H2O(Gibco) were set in TECAN EVO150 Labware stage. The program of “6_RNaseONE_and PURIFICATION” was run. The purified 96 samples were collected to new 96 well plate. Sample concentration: 40 µl of cDNA sample in 96 well plate was concentrated to 12 µl by Speed Vac. 1 µl of aliquot was used for the quality control by measuring concentration (Oligreen) and qPCR respectively.