| Description |
Tumour-associated macrophages (TAM) from LLC-bearing mice treated by V&ssiCtrl and V&ssiLTBR were sorted by FACS AriaIII flow cytometer (BD Immunocytometry Systems). Total RNA of TAM was isolated by using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's procedure. The RNA amount and purity of each sample was quantified using NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA). The RNA integrity was assessed by Bioanalyzer 2100 (Agilent, CA, USA) with RIN number >7.0, and confirmed by electrophoresis with denaturing agarose gel. Poly (A) RNA is purified from 1μg total RNA using Dynabeads Oligo (dT)25-61005 (Thermo Fisher, CA, USA) using two rounds of purification. Then the poly(A) RNA was fragmented into small pieces using Magnesium RNA Fragmentation Module (NEB, cat.e6150, USA) under 94C 5-7min.Then the cleaved RNA fragments were reverse-transcribed to create the cDNA by SuperScript II Reverse Transcriptase (Invitrogen, cat. 1896649, USA), which were next used to synthesise U-labeled second-stranded DNAs with E. coli DNA polymerase I (NEB, cat.m0209, USA), RNase H (NEB, cat.m0297, USA) and dUTP Solution (Thermo Fisher, cat.R0133, USA). An A-base is then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Each adapter contains a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Single- or dual-index adapters are ligated to the fragments, and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme (NEB, cat.m0280, USA) treatment of the U-labeled second-stranded DNAs, the ligated products are amplified with PCR by the following conditions: initial denaturation at 95C for 3 min; 8 cycles of denaturation at 98C for 15 sec, annealing at 60C for 15 sec, and extension at 72C for 30 sec; and then final extension at 72C for 5 min. The average insert size for the final cDNA library was 300±50 bp. At last, we performed the 2x150bp paired-end sequencing (PE150) on an illumina Novaseq 6000 (LC-Bio Technology CO., Ltd., Hangzhou, China). |
