RNA isolation. RNA was isolated using TRIZOL-reagent (ambion cat# 15596018) and DIRECT-ZOL RNA mini-prep kit (cat# 11-330MB). Poly-A RNA isolation. Use 0.2 Total RNA as starting material for ideal mapping efficiency and minimal clonality.  Collect 10 μL oligo (dT) (NEB cat# S1419S) beads per RNA sample. Beads were washed twice with 1x DTBB (20mM Tris-HCl pH7.5, 1M LiCl, 2mM EDTA, 1% LDS, 0.1% Triton X-100). Beads were resuspended in 50uL of 2x DTBB.  50uL of beads were mixed with 50uL RNA and Heated to 65°C for 2 min. RNA-beads were then incubated for 10 min at RT while rotating. RNA-beads were then collected on a magnet and washed 1x each with RNA WB1 (10mM Tris-HCl pH7.5, 0.12 M LiCl, 1mM EDTA, 0.1% LDS, 0.1% Triton X-100)and WB3 (10mM Tris-HCl pH7.5, 0.5M LiCl, 1mM EDTA). Add 50uL Tris-HCl pH7.5 and heat to  80°C for 2 min to elute. Collect RNA and perform a second Oligo-dT bead collection. After washing the second collection, instead of eluting was 1X with 1X First strand buffer (250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2 [ThermoFisher SSIII kit Cat# 18080093). Fragmentation. Then Add 10uL of 2X First strand buffer plus 10mM DTT and fragment DNA at 94°C for 9 min. Collect beads on magnet and transfer eluate containing fragmented mRNA to a new PCR strip.  Should recover 10 μL fragmented RNA. First strand synthesis. We mixed fragmented RNA with 0.5uL Random Primer (3 μg/μL) Life Tech #48190-011, 0.5uL oligo-dT (50uM from SSIII kit), 1uL dNTPs (10mM Life Tech, cat 18427088) and 0.5uL SUPERase-In (ThermoFisher Cat#AM2696) and heat 50°C for 1 min. Immediately place on ice. We then added 5.8uL ddH2O, 0.1 uL Actinomycin (2ug/uL Sigma cat#A1410), 1uL DTT (100mM Life Tech cat# P2325), 0.2uL of 1% Tween and 0.5 uL of Superscript III and incubate 25°C for 10 min, then 50°C for 50 min. Bead clean up. We added 36 μL of RNAClean XP (ampure XP) and mixed, incubating for 15 min on ice. The beads were then collected on a magnet and washed 2X with 75% ethanol. Beads were then air-dried for 10 min and elute with 10 μL nuclease free H2O. Second strand synthesis. 10uL of cDNA/RNA was mixed with 1.5uL 10X Blue Buffer (Enzymatics cat# B0110L), 1uL dUTP/dNTP mix (10mM Affymatrix cat# 77330), 0.1uL dUTP (100mM Affymatrix cat# 77206), 0.2uL RNase H (5U/uL Enzymatics cat# Y9220L), 1uL DNA polymerase I (10U/uL Enzymatics cat#P7050L), 0.15 uL 1% Tween-20 and 1.05uL nuclease free water. Reaction was incubated at 16°C for 2.5 hours. Bead clean up. DNA was purified by adding 1uL Seradyn “3 EDAC” SpeedBeads (Thermo 6515-2105-050250) per reaction in 28uL 20% PEG8000/2.5 M NaCl (13% final concentration) and incubating at RT for 10min. Beads were then collected on a magnet and washed 2X with 80% Ethanol. Beads were air-dried for 10min and eluted in 40uL of nuclease free water. DNA is ready for library prep. dsDNA End Repair. We mixed 40uL of DNA from ChIP or RNA protocols with 2.9uL of H2O, 0.5uL 1% Tween-20, 5uL 10X T4 ligase buffer (Enzymatics cat# L6030-HC-L), 1uL dNTP mix (10 mM  Affymetrix 77119), 0.3 uL T4 DNA pol (Enzymatics P7080L), 0.3uL T4 PNK (Enzymatics Y9040L), 0.06uL Klenow (Enzymatics P7060L) and incubated for 30min at 20°C. 1uL of Seradyn “3 EDAC” SpeedBeads (Thermo 6515-2105-050250) in 93 μL 20% PEG8000/2.5 M NaCl (13% final) was added and incubated for 10 min. Bead clean-up. Beads were collected on a magnet and washed 2X with 80% ethanol. Beads were air-dried for 10 min and then eluted in 15uL ddH2O. dA-Tailing. DNA was mixed with 10.8uL ddH2O, 0.3uL 1% Tween-20, 3uL Blue Buffer (Enzymatics cat# B0110L), 0.6uL dATP (10mM Tech 10216-018), 0.3uL Klenow 3'- 5' Exo (Enzymatics P7010-LC-L) and incubated for 30min at 37°C. 55.8uL 20% PEG8000/2.5 M NaCl (13% final) was added an incubated for 10 min. Then bead clean up was done. Beads were eluted in 14uL. Y-Shape Adapter Ligation. Sample was mixed with 0.5uL of a BIOO barcode adapter (BIOO Scientific cat# 514104), 15uL Rapid Ligation Buffer (Enzymatics cat@ L603-LC-L), 0.33uL 1% Tween-20 and 0.5uL T4 DNA ligase  HC (Enzymatics L6030-HC-L) and incubated for 15 min at RT.  7 μL of 20% PEG8000/2.5 M NaCl was added and incubated for 10min at RT. Bead clean up was performed and beads were eluted in 21uL. 10uL was then used for PCR amplification (14 cycles) with IGA and IGB primers (AATGATACGGCGACCACCGA, CAAGCAGAAGACGGCATACGA).
GEO Accession
GSM3041217