GSM8827804: VASA-WEE11-DMSO-r2; Homo sapiens; RNA-Seq
RPF: Cells were collected by trypsinization (TrypLE, Gibco), counted, washed in PBS0, and approximately 2000 were resuspended in 2.5 µL polysome lysis buffer [20 mM Tris pH 7.5, 15 mM MgCl2, 5 mM CaCl2, 150 mM NaCl, 1% Triton X-100, 2 U/µL RNaseIN Plus (Promega), 0.1 mM Cycloheximide (Sigma Aldrich), 0.1 mM Chloramphenicol (Sigma Aldrich)] and incubated on ice. No RNA purification was performed.  VASA: Cells were collected by trypsinization (TrypLE, Gibco), counted, washed in PBS0, and approximately 2000 were resuspended in 2.5 µL of 2.5 µM SORT-seq primer, snap frozen, and stored at -80 °C until processing. No RNA purification was performed. RPF: To generate ribosome-protected footprints, 2.5 µL of Micrococcal Nuclease (MNase, 62.5 U/µL, New England Biolabs) was added to each sample and incubated at 37 °C for 30 minutes. In order to stop digestion, 2.5 µL of stop mix [0.0186 U/µL Thermolabile Proteinase K (New England Biolabs), 62 mM EGTA (Sigma Aldrich), 16.5 mM EDTA (Ambion), and 697.5 mM guanidium thiocyanite (GuSCN, Sigma Aldrich)] was added to each well, and samples were incubated at 37 °C for 30 minutes then 55 °C for 10 minutes and held at 4 °C.  After ribosome footprint digestion, libraries were constructed using a one-pot small-RNA library preparation protocol that incorporated end repair, two RNA ligations, cDNA synthesis, and an indexing PCR. First, 2.5 µL of end-repair mix [4.1× of 10× T4 RNA Ligase Buffer (New England Biolabs), 16.4 mM MgCl2, 4.1 mM uridine triphosphate (New England Biolabs), 1.37 U/µL T4  Polynucleotide Kinase (New England Biolabs), and 0.82 U/µL RNaseIN Plus] was added to each well, and plates were incubated at 37 °C for 1 hour and held at 4 °C. Next, 13.2 µL of 3' ligation brew [1× T4 RNA Ligase Buffer (New England Biolabs), 1 µM pre-adenylated 3' adapter (OMV630 Integrated DNA Technologies), 35.5 % PEG-8000 (New England Biolabs), 0.1 % Tween-20 (Sigma Aldrich), 1 U/µL RNaseIN Plus, and 21.3 U/µL T4 RNA Ligase 2 Truncated KQ (New England Biolabs)] was added to each well and plates were incubated at 4 °C for 18 hours. The cDNA synthesis primer was then pre-annealed to the 3' ligation products by adding 2.5 µL of the RT primer mix [5.2 µM RT primer (OMV572, Integrated DNA Technologies), 13.5 µM adenosine triphosphate (ATP, New England Biolabs), and 1 % Tween-20] to each well, heating to 65 °C for 1 minute, 37 °C for 2 minutes, 25 °C for 2 minutes, and holding at 4 °C. Five-prime adapters were then ligated by adding 7.8 µL of 5' ligation brew [1× T4 RNA Ligase Buffer, 30.75 % PEG-8000, 0.1 % Tween-20, 0.5 µM 5' adapter (OMV632, Integrated DNA Technologies), 1.25 U/µL T4 RNA Ligase 1 (Ambion)] and incubating at 37 °C for 2 hours and holding at 4 °C. Complementary DNA synthesis was then peformed by adding 38.5 µL of reverse transcription brew [1.88× 5× RT Buffer (ThermoFisher Scientific), 1.25 mM dNTPs (Promega), 0.1875 % Tween-20, 1.875 U/µL RNaseIN Plus, and 9.375 U/µL Maxima H Minus Reverse Transcriptase (ThermoFisher Scientific)] to each well, and heating at 50 °C for 1 hour, then 85 °C for 5 minutes and holding at 4 °C. Finally, libraries were indexed during PCR by first transferring 3.6 µL of 20 µM unique forward index primers (Integrated DNA Technologies) and 78 µL of PCR brew [1.5× Q5 Hot Start High-Fidelity 2× Master Mix (New England Biolabs), 0.15 % Tween-20, and 0.94 µM reverse index primer (Integrated DNA Technologies)] to half of the cDNA from each sample. Plates were then incubated at 98 °C for 30 s followed by 8 cycles of 98 °C for 15 s, 65 °C 30 s, 72 °C for 30 s, and then a final incubation at 72 °C for 5 min and holding at 4 °C. Plates were then frozen at -20 °C until gel purification.  After library construction the samples were purified. Product was cleaned up using AMPure XP beads (Beckman Coulter) that had been diluted 6× in bead binding buffer [20 % PEG-8000 (Sigma Aldrich) 2.5 M Sodium Chloride (Sigma Aldrich)]; diluted beads were added to the sample at a 2.1:1 ratio, and the final product was resuspended in 20 µL of low TE buffer [LoTET, 3 mM Tris-HCl pH 8.0 (Ambion), 0.2 mM EDTA pH 8.0 (Gibco), 0.1 % Tween-20]. Half of each of the cleaned-up library pools was then run on a 20 cm 7 % polyacrylamide gel at 300 V until the lower marker ran off of the gel. The ~10 base-pair region from 175-185, corresponding to an insert size of ~30-40 nt was excised. The band was then crushed and soaked overnight at 4 °C in LTET8. Finally, eluate was cleaned up using AMPure XP beads that had been diluted 6× in bead binding buffer and resuspended in LTET8.  VASA: Cells were first lysed by adding 2.5 µL of Lysis and Fragmentation brew [3.4× First Strand Buffer (Invitrogen), 0.024 U/µL Thermolabile Proteinase K (New England Biolabs), 0.2 % Triton X-100, 1:125000 diluted ERCC Spike-in] and incubating at 25 °C for 1 hour, then 55 °C for 10 minutes and holding at 4 °C. Next, 2.5  µL of End Repair and Poly(A) tailing brew was added [0.6× First Strand Buffer (Invitrogen), 20 mM DTT (Invitrogen), 7.5 µM ATP (New England Biolabs), 0.075 U/µL E. coli Poly(A) Polymerase (New England Biolabs), 1 U/µL T4 Polynucleotide Kinase (New England Biolabs), 4 U/µL RNase OUT (Invitrogen)] and incubated at 37 °C for 1 hour and held at 4 °C. Then 2.5  µL of Reverse Transcription Brew was added [2 mM each dNTP (Promega), 16 U/µL SuperScript III (Invitrogen)] and incubated at 50 °C for 1 hour and held at 4 °C. Finally, 55 µL of Second Strand brew [1.14× Second Strand Buffer (Invitrogen), 0.23 mM each dNTP (Promega), 0.32 U/µL E. coli DNA Polymerase I (Invitrogen), 0.018 U/µL RNase H (Invitrogen)] was added and samples were incubated at 16 °C, 85 °C for 20 minutes, and held at 4 °C. Double stranded libraries were cleaned up using AMPure XP beads (Beckman) that had been diluted 8× in Bead Binding Buffer [20 % PEG-8000 (Sigma Aldrich), 2.5 M NaCl (Ambion), 0.02 % Sodium Azide (Sigma Aldrich)], resuspended in 12 µL of water, and amplified using in vitro transcription at 37 °C overnight using the MegaScript T7 kit (Invitrogen) in a 30 µL reaction.  In vitro transcription was stopped by adding 12 µL of ExoSAP-IT (Invitrogen) and incubating at 37 °C  for 15 minutes and holding at 4 °C. Resulting amplified RNA (aRNA) was then cleaned up using AMPure XP Beads at a 1.8× ratio and resuspended in 10 µL of water. 600 ng of aRNA was then mixed with 10 µL hybridization brew [100 mM Tris pH 7.5, 200 mM NaCl (Ambion), 5 µM rRNA depletion oligonucleotides], incubated at 95 °C for 2 minutes, then decreased to 45 °C at a rate of 0.1 °C/s. While keeping samples at 45 °C, 10 µL of RNase H mix [100 mM Tris pH 7.5, 200 mM NaCl (Ambion), 40 mM MgCl2, 1 U/µL Hybridase Thermostable RNase H (Epicenter)] was added, mixed by pipetting, and incubated at 45 °C for 30 minutes and held at 4 °C. Next, 30 µL of DNase mix [1.67 mM CaCl2, 0.133 U/µL RQ1 DNase (Promega)] was added and samples were incubated at 37 °C for 30 minutes. The resulting rRNA-depleted samples were cleaned up using AMPure XP beads at a 1.6× ratio and eluted in 5 µL of water.  The cleaned up, rRNA-depleted samples were then each mixed with 1 µL of RA3 adapter (/5rApp/TGGAATTCTCGGGTGCCAAGG/3ddC/, 20 µM), denatured at 70 °C for 2 minutes, then snap chilled on ice. 4 µL of ligation brew was added [2.5× T4 RNA Ligase Reaction Buffer (New England Biolabs), 50 U/µL T4 RNA Ligase 2 truncated (New England Biolabs), 10 U/µL RNaseOUT (Invitrogen), 12.5% PEG-8000 (New England Biolabs)], and incubated at 25 °C for 1 hour and held at 4 °C. Ligated RNA was then converted to cDNA by adding 3 µL of dNTP-primer mix [3.33 µM each dNTPs (Promega), 13.33 µM RTP (CAAGCAGAAGACGGCATACGA)], incubating at 65 °C for 5 minutes and snap chilling on ice. 8 µL of RT brew [2.5× First Strand Buffer (Invitrogen), 12.5 mM DTT (Invitrogen), 5 U/µL RNaseOUT (Invitrogen), 25 U/µL SuperScript III (Invitrogen)], was added, incubated at 50 °C for 1 hour, 70 °C for 15 minutes, and held at 4 °C. RNA was next degraded by adding 1 µL RNase A (10 mg/mL, ThermoFisher Scientific) and incubating at 37 °C for 30 minutes. Complementary DNA was cleaned up using AMPure XP beads at a 1× ratio and resuspended in 10 µL of water. Purified cDNA was amplified by adding PCR brew [1.25 × NEBNext PCR mix (New England Biolabs), 0.5 µM RP1 (AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA), and 0.5 µM indexed RPI (CAAGCAGAAGACGGCATACGAGAT[6 bases]GTGACTGGAGTTCCTTGGCACCCGAGAATTCCA)], and incubating at 98 °C for 30 s followed by 8 cycles of 98 °C for 15 s, 60 °C 30 s, 72 °C for 30 s, and then a final incubation at 72 °C for 5 min and holding at 4 °C. Amplified and indexed libraries were cleaned up with two successive AMPure XP bead cleanups with 0.8× ratio and resuspended in LTET8.