| Experimental protocol | For ESCs and NPCs, cell pellets were resuspended in 1 mL of TriPure and RNA extracted following standard protocol. DNA was digested with Turbo DNase-I (Invitrogen, Cat. No AM2238) at 37°C for 30 min, RNA was re-purified using TriPure, and resuspended in modBTE (10mM Bis/Tris pH 6.7, 0.1 mM EDTA). For mouse tissues, brains, and testes in TriPure were homogenized using glass beads and clarified by centrifugation at 12,000g for 1 min. RNA was purified as for ESCs and NPCs. For mouse sperm, total RNA was extracted using the same method previously described for epididymosomes.89 Briefly, sperm were resuspended in 120 μL of water and 66 μL of sperm lysis buffer were added [120mM Tris-HCl (pH 8), 6.4 M Guanidine-HCl, 5% Tween-20, 5% Triton-X-100, 120 mM EDTA]. Proteins were digested by adding 6.6 μL of 20 mg/mL proteinase K and 6.6 μL of 1M DTT, followed by incubation at 60°C for 15 min under 600 rpm constant shaking. Volume was adjusted with water to 400 μL and 400 μL of TriPure were added. RNA was extracted by adding 120 μL of BCP phase separation reagent (Molecular Research Center, Cat. No BP151) and precipitated with isopropanol. DNA was digested with Turbo DNase-I at 37°C for 30 min, RNA was re-purified using TriPure, and resuspended in modBTE. RNA integrity was verified on 1.5% formaldehyde/agarose gels. For enrichment of RNAs < 200 nts, the Zymo RNA Clean and Concentrator-5 kit (Zymo Research, Cat. No R1013) was used, starting from 1 μg of total RNA. For enrichment of RNAs < 50 nts, 18 μg of total RNA were run on a denaturing 12% polyacrylamide-urea gel, and the section between 10 and 50 nt markers was excised. Gel pieces were shredded trough pierced 0.5 mL tubes, and RNA was eluted from the gel by overnight incubation in 400 μL RNA elution buffer (10mM Bis/Tris pH 6.7, 300 mM NaCl, 10 mM EDTA) at 4°C with constant rotation. Eluate was filtered through 5 μm PVDF spin filters (EMD Millipore, Cat. No UFC30SV00), and RNA was precipitated by adding 1 μL glycoblue (Invitrogen, Cat. No AM9516), 40 μL 3 M NaOAc pH 5.2, and 1.1 mL of ice-cold 100% EtOH, followed by incubation at -80°C for 1 h. RNA was pelleted at 20,000 g for 30 min at 4°C, washed once with 1 mL of 70% EtOH, once with 1 mL of 80% EtOH, air-dried for 5 min, and resuspended in modBTE buffer. rRNA depletion with the NEB rRNA depletion Kit (New England Biolabs, Cat No E7400S) was performed starting from 1 μg of total ESC RNA and following the standard protocol, with the exception that rRNA-depleted RNA was purified using TriPure instead of SPRI. rRNA depletion with RiboCop (Lexogen, Cat. No 144.24) was performed starting from 500 ng of total ESC RNA and following the standard protocol, except that the rRNA-depleted RNA were first subjected to Turbo DNase digestion (to remove DNA probes) and purified using TriPure. rRNA depletion with the ZapR module of the SMARTer Stranded Total RNA-Seq Kit v3 - Pico Input Mammalian (Takara, Cat No 634485) starting from indexed LIDAR libraries made from 100 ng of total RNA input and following the standard protocol (12 cycles of final PCR). The desired amount of total and fractionated RNAs was diluted to 1 μL and mixed with 0.4 μL of 10 μM LIDAR_RT_primer. To anneal the LIDAR_RT_primer to the template RNA, samples were heated to 65°C for 5 min, then cooled to 4°C (0.5°C/s). To initiate RT, 6.28 μL of RT_mix [25 mM Tris-HCl pH 8.3, 20 mM NaCl, 2.5 mM MgCl2, 8 mM DTT, 5% PEG-8000, 0.5 mM dNTPs, 1 mM GTP, 0.5 U/μL RNase inhibitor, murine (New England Biolabs, Cat. No M0314S), and 2U/μL Maxima H-minus RT (Thermo Scientific, Cat. No EP0752); concentrations refer to a final volume of 8 μL] were added and samples were incubated at 25°C for 10 min. To further promote cDNA synthesis and template switch, temperature was raised to 42°C, 0.32 μL of 50 μM LIDAR_TSO_mix were added, and samples were incubated at 42°C for 80 min, followed by 10 cycles at 50°C for 2 min and 42°C for 2 min, then at 85°C for 5 min. To pre-amplify and add adapters to cDNA, 12 μL of KAPA_mix [1X KAPA HiFi HotStart buffer Ready Mix (Roche, Cat. No KK2601), 1 μM LIDAR_preamp_f, and 0.1 μM LIDAR_preamp_r; concentrations refer to a final volume of 20 μL] were added and samples were incubated with the following PCR cycling conditions: denaturation (95°C for 3 min), 18 x (98°C for 20 s, 70°C for 30 s, 72°C for 30 s), final extension (72°C for 5 min). Quality of adapter-containing libraries was assessed by loading 5 μL on a 2% agarose gel. To generate the final libraries, 2 μL of pre-amplified were diluted to 37 μL with 10mM Tris-HCl pH 8, 0.5 μL of each 10 μM custom Nextera indexing primers was added, followed by 12 μL of Q5_mix [1X Q5 buffer, 0.5 mM dNTPs, 0.02 U/μL Q5 High-Fidelity DNA Polymerase (New England Biolabs, Cat. No M0491); concentrations refer to a final volume of 50 μL]. Samples were incubated with the following PCR cycling conditions: denaturation (98°C for 30 sec), 18 x (98°C for 10 s, 65°C for 20 s, 72°C for 20 s), final extension (72°C for 2 min). Indexed libraries were purified using 2.3X SPRI beads (Beckman Coulter, Cat. No B23319) and eluted in 50 μL of TE buffer (10 mM Tris-HCl pH 8, 1 mM EDTA).For 3′-LIDAR, the LIDAR-3_RT_oligo was generated by mixing equimolar amounts of LIDAR_RT_primer and LIDAR-3_RT_antisense followed by heating at 95°C for 5 min, and a slow cool down to 25°C (0.1°C/s). To prepare 3′-LIDAR libraries, 1 μL of input RNA was first denatured at 70°C for 2 min, then temperature was lowered to 50°C and 0.4 μL of 1 μM LIDAR-3_RT_oligo were added. Samples were incubated at 50°C for further 2 min, then temperature was lowered to 4°C (0.5°C/s). To initiate RT, 6.28 μL of RT_mix were added and samples were incubated at 25°C for 10 min. To further promote cDNA synthesis and template switch, temperature was raised to 42°C, 0.32 μL of 50 μM LIDAR_TSO_mix were added, and samples were incubated at 50°C for 80 min, followed by 10 cycles at 55°C for 2 min and 50°C for 2 min, then at 85°C for 5 min. LIDAR-3_RT_antisense was digested by adding 1 μL of USER II enzyme (New England Biolabs, Cat. No M5508S) and incubating at 37°C for 30 min. USER II was heat inactivated by incubation at 65°C for 10 min. To pre-amplify and add adapters to cDNA, 12 μL of KAPA_mix (1X KAPA HiFi HotStart buffer ready mix, 2 μM LIDAR_preamp_f, and 0.5 μM LIDAR_preamp_r; concentrations refer to a final volume of 20 μL) were added and samples were incubated with the following PCR cycling conditions: denaturation (95°C for 3 min), 6x (98°C for 20 s, 63°C for 30 s, 72°C for 30 s), 12x (98°C for 20 s, 72°C for 50 s), final extension (72°C for 5 min). Quality of adapter-containing libraries was assessed by loading 5 μL on a 2% agarose gel. To generate the final libraries, 2 μL of pre-amplified libraries were diluted to 37 μL with 10 mM Tris-HCl pH 8, 1 μL of each 10 μM custom Nextera indexing primers was added, followed by 12 μL of Q5_mix (1X Q5 buffer, 0.5 mM dNTPs, 0.02 U/μL Q5 high-fidelity DNA polymerase; concentrations refer to a final volume of 50 μL]. Samples were incubated with the following PCR cycling conditions: denaturation (98°C for 30 sec), 7x (98°C for 10 s, 65°C for 20 s, 72°C for 20 s), final extension (72°C for 5 min). Indexed libraries were purified using 2.4X SPRI beads and eluted in 50 μL of TE buffer (10 mM Tris-HCl pH 8, 1 mM EDTA). LIDAR and 3′-LIDAR libraries were analyzed on a 2% agarose gel and quantified using NEBNext Library Quant Kit for Illumina (New England Biolabs, Cat. No E7630L). Libraries were sequenced on Illumina NextSeq500 or NextSeq1000 instruments. |
