title: GSM4061795: AM_LPS02h_ipB161219; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was isolated from in vitro culture cells and purified using a Direct-zol RNA MicroPrep Kit (Zymo Research) according to the manufacturer's instructions (Zymo Research). FACS purified cells were pelleted and put into 150 μl lysis/Oligo d(T) Magnetic Beads binding buffer (100mM Tris-HCl pH7.5, 500mM LiCl, 10mM EDTA pH8.0, 1% LiDS, 5mM DTT) and stored at –80°C until processing. mRNAs were enriched by incubation with Oligo d(T) Magnetic Beads (NEB, S1419S). Sequencing libraries were prepared according to the Smart-seq2 method45 with some modifications. 500-5000 FACS isolated cells in Trizol (Thermo Fisher) were used as starting material. RNA was extracted with the Direct-zol MicroPrep kit (Zymo Research) with on-column DNAseI treatment. 10 µL purified RNA was mixed with 5.5 µL of SMARTScribe 5X First-Strand Buffer (Clontech), 1 µL polyT-RT primer (2.5 µM, 5'-AAGCAGTGGTATCAACGCAGAGTAC(T30)VN, 0.5 µL SUPERase-IN (Ambion), 4 µL dNTP mix (10 mM, Invitrogen), 0.5 µL DTT (20 mM, Clontech) and 2 µL Betaine solution (5 M, Sigma), incubated 50C 3 min. 3.9 µL of first strand mix, containing 0.2 µL 1% Tween-20, 0.32 µL MgCl2 (500 mM), 0.88 µL Betaine solution (5 M, Sigma), 0.5 µL (5 M, Sigma) SUPERase-IN (Ambion) and 2 µL SMARTScribe Reverse Transcriptase (100 U/µL Clontech) was added and incubated one cycle 25C 3 min., 42C 60 min. 1.62 µL template switch (TS) reaction mix containing 0.8 µL biotin-TS oligo (10 µM, Biotin-5'-AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3'), 0.5 µL SMARTScribe Reverse Transcriptase (100 U/µL Clontech) and 0.32 µL SMARTScribe 5X First-Strand Buffer (Clontech) was added, then incubated at 50C 2 min., 42C 80 min., 70C 10 min. 14.8 µL second strand synthesis, pre-amplification mix containing 1 µL pre-amp oligo (10 µM, 5'AAGCAGTGGTATCAACGCAGAGT-3'), 8.8 µL KAPA HiFi Fidelity Buffer (5X, KAPA Biosystems), 3.5 µL dNTP mix (10 mM, Invitrogen) and 1.5 µL KAPA HiFi HotStart DNA Polymerase (1U/µL, KAPA Biosystems), was added,  then amplified by PCR: 95C 3 min., 8 cycles 98C 20 seconds, 67C 15 sec and 72C 6 min, final extension 72C 5 min.  The synthesized dsDNA was purified using Sera-Mag Speedbeads (Thermo Fisher Scientific) with final 8.4% PEG8000, 1.1M NaCl, then eluted with 13 µL UltraPure water (Invitrogen). The product was quantified by Qubit dsDNA High Sensitivity Assay Kit (Invitrogen) and libraries were prepared using the Nextera DNA Sample Preparation kit (Illumina). Tagmentation mix containing 11 µL 2X Tagment DNA Buffer and 1 µL Tagment DNA Enzyme was added to 10 µL purified DNA, then incubated at 55C 15 min. 6 µL Nextera Resuspension Buffer (Illumina) was added and incubated at room temperature for 5 min. Tagmented DNA was purified using Sera-Mag Speedbeads (Thermo Fisher Scientific) with final 7.8% PEG8000, 0.98M NaCl, then eluted with 25 µL UltraPure water (Invitrogen). Final enrichment amplification was performed with Nextera primers,  adding 1 µL  Ad1 (100 µM, universal no barcode), 1 µL unique Ad2.n (100 µM, N7xx) barcoding primers and 27 µL NEBNext High-Fidelity 2X PCR Master Mix (New England BioLabs), then amplified by PCR: 72C 5 min., 98C 30 sec., 8-13 cycles 98C 10 seconds, 63C 30 sec., and 72C 1 min. Libraries were purified using 1.5 volumes of SpeedBeads in 2.5M NaCl, 20% PEG8000, further size selected using 10% PAGE/TBE gels, eluted and quantified with Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific), pooled and sequenced on a NextSeq 500 (Illumina) for 76 cycles at a depth of 25 to 30 million single end reads per sample.
identifiers.namespace: GEO
identifiers.id: GSM4061795