GSM8265669: NEB, total, testis, b2; Mus musculus; RNA-Seq
LIDAR and LIDAR-3 library preparation: The desired amount of total and fractionated RNAs was diluted to 1μl and mixed with 0.4μl of 10μM LIDAR_RT_primer (/5Biosg/GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGNNNNND). To anneal the LIDAR_RT_primer to the template RNA, samples were heated to 65°C for 5 min, then temperature was lowered to 4°C (0.5°C/s). To initiate retrotranscription, 6.28μl of RT_mix [25mM Tris-HCl pH=8.3, 20mM NaCl, 2.5mM MgCl2, 8mM DTT, 5% PEG-8000, 0.5mM dNTPs, 1mM GTP, 0.5 U/μl Murine RNase inhibitor, and 2U/μl Maxima H-minus Reverse Transcriptase – 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, 0.32μl of 50μM LIDAR_TSO_mix (equimolar ratio of the following oligos: /5Biosg/CGTCAGATGTGTATAAGAGACAGtgNNcgNNagNNrGrGrG, /5Biosg/CGTCAGATGTGTATAAGAGACAGvhNNcgNNagNNrGrGrG, /5Biosg/CGTCAGATGTGTATAAGAGACAGvhgNNcgNNagNNrGrGrG, /5Biosg/CGTCAGATGTGTATAAGAGACAGvhtgNNcgNNagNNrGrGrG, /5Biosg/CGTCAGATGTGTATAAGAGACAGvhmtgNNcgNNagNNrGrGrG) 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, 12μl of KAPA_mix [1X KAPA HiFi HotStart buffer Ready Mix, 0.5μM LIDAR_preamp_f (TCGTCGGCAGCGTCAGATGTGTATAAGAGAC*A*G), and 0.1μM LIDAR_preamp_r (GTCTCGTGGGCTCGGAGA) – 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 cycles of amplification (98°C for 20s, 70°C for 30s, 72°C for 30s), final extension (72°C for 5 min). Quality of adapter-containing libraries was assessed by loading 5ul on a 2% agarose gel. To generate the final libraries, 2μl of pre-amplified were diluted to 37μl with 10mM Tris-HCl pH=8, 0.5μl of each 10μM custom Nextera indexing primers was added, followed by 12μl of Q5_mix [1X Q5 buffer, 0.5mM dNTPs, 0.02 U/µl Q5 High-Fidelity DNA Polymerase (Cat. No M0491, New England Biolabs) – 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 cycles of amplification (98°C for 10s, 65°C for 20s, 72°C for 20s), final extension (72°C for 2 min). Indexed libraries were purified using 2.3X SPRI beads and eluted in 50μl of TE buffer (10mM Tris-HCl pH=8, 1mM EDTA). For LIDAR-3, the LIDAR-3_RT_oligo was generated by mixing equimolar amounts of LIDAR-3_RT_sense (/5Biosg/GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGNNNNNN) and LIDAR-3_RT_antisense (CdUGdUCdUCdUdUAdUACACAdUCdUCCGAG/3ddC/), followed by heating at 95°C for 5 min, and a slow cool down to 25°C (0.1°C/s). To prepare LIDARsp libraries, 1μl of input RNA was first denatured at 70°C for 2 min, then temperature was lowered to 50°C and 0.4μl of 1μM LIDAR-3_RT_oligo were added. Samples were incubated at 50°C for further 2min, then temperature was lowered to 4°C (0.5°C/s). To initiate retrotranscription, 6.28μl 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, 0.32μl of 50μM 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 1ul of USER II enzyme 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, 12μl of KAPAsp_mix [1X KAPA HiFi HotStart buffer Ready Mix, 2μM LIDAR_preamp_f (TCGTCGGCAGCGTCAGATGTGTATAAGAGAC*A*G), and 0.5μM LIDAR_preamp_r (GTCTCGTGGGCTCGGAGA) – 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), 6 cycles of 3-step amplification (98°C for 20s, 63°C for 30s, 72°C for 30s), 12 cycles of 2-step amplification (98°C for 20s, 72°C for 50s), final extension (72°C for 5 min). Quality of adapter-containing libraries was assessed by loading 5ul on a 2% agarose gel. To generate the final libraries, 2μl of pre-amplified were diluted to 37μl with 10mM Tris-HCl pH=8, 1μl of each 10μM custom Nextera indexing primers was added, followed by 12μl of Q5sp_mix [1X Q5 buffer, 0.5mM 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), 7 cycles of amplification (98°C for 10s, 65°C for 20s, 72°C for 20s), final extension (72°C for 5 min). Indexed libraries were purified using 2.4X SPRI beads and eluted in 50μl of TE buffer (10mM Tris-HCl pH=8, 1mM EDTA). Small RNA-seq and mRNA-seq library preparation Small RNA libraries were prepared using the NEBnext Small RNA Library Prep Set for Illumina (Cat. No E7330S, New England Biolabs), following the standard protocol with the following parameters: 1) 1:2 dilution of 3'SR Adaptor, SR RT Primer, and 5'SR Adaptor 2) 15 indexing PCR cycles 3) cleanup of final libraries with QIAgen MinElute PCR purification kit, followed by a second cleanup with 2X SPRI. Libraries were analyzed on a 6% non-denaturing polyacrylamide gel. For mRNA-seq libraries, polyA RNA was enriched from 2 μg total RNA using Dynabeads Oligo(dT)25. Poly-A-enriched fraction was used for library construction using the NEBnext Ultra II Directional Library Prep Kit for Illumina (Cat. No. E7760L, New England Biolabs), following the standard protocol but using half the reaction volumes recommended. Libraries were analyzed on a 2% agarose gel. Both small RNA-seq and mRNA-seq libraries were quantified using NEBNext Library Quant Kit for Illumina (Cat. No E7630L, New England Biolabs). Libraries were sequenced on an Illumina NextSeq500 instrument.