GSM3052359: F480Hi_HFD_rep2; Mus musculus; RNA-Seq
NCD and HFD mice were euthanized, and freshly-prepared collagenase P (Roche, Indianapolis, IN) solution (0.5 mg/ml) was injected into the pancreas via the common bile duct. The perfused pancreas was digested at 37°C for 20 min, and the islets were handpicked under a stereoscopic microscope, and then dispersed into single cell suspensions. Macrophages were labeled with mAbs against CD11b, CD11c, F4/80 and sorted using FACSAria II (BD, San Jose, CA). Sequencing libraries were prepared according to the Smart-seq2 method (Picelli, et al. 2013) with some modifications. 3000-5000 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 of 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-3'), 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 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 Index 1 primers (100 µM, N7xx), 1 µL Index 2 primers (100 µM, N5xx) 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 size selected, quantified 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 40 million single end reads per sample.
GEO Accession
GSM3052359