Ampure beads

For RNA-seq analysis, total RNA was extracted from Myo-EVs, primary mouse bone marrow cells and osteoblasts with TRIZOL reagent (Thermo Fisher Scientific, Inc) and assessed by an Agilent Bioanalyzer with the RNA 6000 Pico Kit (Agilent, Santa Clara, CA, USA). Sequencing library was prepared using the NEBNext Small RNA Library Prep Kit (E7330, New England Biolabs, Ipswich, MA, USA). Total RNA (90 ng) was ligated to 3’ Adaptor, and excess adaptor was subsequently absorbed. RNA fragments were further ligated to 5’ Adaptor for cDNA synthesis. Synthesized cDNA was amplified by PCR for 12 cycles. Small-RNA library was size-selected by AMPure beads (NC9933872, Thermo Fisher Scientific, Inc) and verified with the Bioanalyzer DNA High-sensitivity Kit (5067–4626, Agilent). For obtaining 2 × 36-base paired-end reads, sequencing was performed by the Illumina NextSeq500 (Illumina, San Diego, CA, USA). FASTQ files were imported to CLC Genomics Workbench (Ver.10.1.1, Qiagen, Germantown, MD, USA). Reads were grouped by sequence and the read groups were matched to miR-base annotated micro RNAs. Raw counts of each sample were converted by total count 1,000,000 at first, and the converted counts were further normalized by scaling method with means as normalization values, median mean as reference and trimming percentage 5 for comparing multiple samples.

Total RNA was extracted with a TaKaRa MiniBEST Plant RNA Extraction Kit and genomic DNA was removed by DNaseI (New England Biolabs). PAT-seq libraries were prepared as previously described with modifications (Lin et al., 2020 (link)). Two micrograms of total RNA were fragmented by 5× first strand buffer (TaKaRa) at 94°C for 4 min. Poly(A) RNAs were then enriched by oligo(dT)₂₅ beads (New England Biolabs). Reverse transcription was performed with oligo d(T)₁₈ primers by SMARTScribe™ Reverse Transcriptase (TaKaRa) for 2 h at 42°C. Then, a modified 5′ adaptor and SMARTScribe Reverse Transcriptase were added for another 2 h at 42°C. The cDNA generated was then purified with AMPure beads and amplified with Phire II (Thermo Fisher Scientific). The amplification products were then separated on a 2% agarose gel and 300–500 bp fragments were purified with a Zymoclean Gel DNA Recovery Kit. The concentration and quality of libraries were tested by a Qubit 2.0 and an Agilent Bioanalyzer 2100, and then sequenced on an Illumina HiSeq 2500 platform with 100-bp rapid sequencing mode.

The Ampure beads (Beckman Coulter Inc., Indianapolis, IN, United States) were used to purify the amplicons of each sample by eliminating the unused primers and to prepare the sequencing libraries an additional 8 cycles of PCR was executed via Illumina barcoded adapters (Supplementary Table 1). Further, prepared libraries were purified by Ampure beads and quantified with the help of a QuDye-dsDNA HS assay kit (Thermo Fisher, Bangalore, India). The Illumina Miseq with a 2 × 300PE v3 sequencing kit was used for sequencing at Biokart India Pvt. Ltd., Bengaluru, India. The raw data was submitted to NCBI Short Read Archive (SRA) under BioProject accession number PRJNA767533.

Lysis plates were prepared by dispensing 4 μl lysis buffer as described in Schaum et al. (2018) (link). After dissociation, single tdTomato⁺ cells were sorted in 96-well plates using SH800S (Sony). Immediately after sorting, plates were sealed with a pre-labelled aluminum seal, centrifuged, and flash frozen on dry ice. cDNA synthesis and library preparation were performed using the Smart-seq2 protocol (Picelli et al., 2014 (link)). Wells of each library plate were pooled using a Mosquito liquid handler (TTP Labtech). Pooling was followed by two purifications using 0.7x AMPure beads (Fisher, A63881). Library quality was assessed using capillary electrophoresis on a Fragment Analyzer (AATI), and libraries were quantified by qPCR (Kapa Biosystems, KK4923) on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). Libraries were sequenced on the NextSeq 500 Sequencing System (Illumina) using 2 × 75 bp paired-end reads and 2 × 8 bp index reads.

Following library preparation, wells of each library plate were pooled using a Mosquito liquid handler (TTP Labtech). Row A of the thymus plates, which contained bulk cells, was pooled separately. Pooling was followed by two purifications using 0.7x AMPure beads (Fisher, A63881). Library quality was assessed using capillary electrophoresis on a Fragment Analyzer (AATI), and libraries were quantified by qPCR (Kapa Biosystems, KK4923) on a CFX96 Touch Real-Time PCR Detection System (Biorad). Plate pools were normalised to 2 nM and sequenced on the NovaSeq 6000 Sequencing System (Illumina) using 2 × 100 bp paired-end reads with an S4 300 cycle kit (Illumina, 20012866). Row A thymus pools were normalised to 2 nM and sequenced separately on the NextSeq 500 Sequencing System (Illumina) using 2 × 75 bp paired-end reads with a High Output 150 cycle kit (Illumina, FC-404–2002).

Each 3C library DNA sample was digested with Dpn II (NEB, R0543S). The 4C ligation was in 10 ml with 50 μl T4 DNA ligase (400 U/μl, NEB) at room temperature overnight. DNA was then ethanol precipitated and purified with the QIAquick PCR purification kit (Qiagen, 28106). The 4C-DpnII library was then amplified on the Veriti 96-Well Thermo Cycler (Applied Biosystems). Limited PCR (round-1) was conducted with the MYC-promoter region as the bait. The PCR products were purified using PCR purification kit and re-amplified by limited PCR (round-2) with primers containing Illumina sequencing adapters (Read 1 and Read 2 adapters, respectively). Primer sequences are in Supplementary Table S1. 4C library purification was performed using Ampure beads (Fisher, NC9959336) and then analyzed by deep sequencing (85 M PE150 reads per sample) using the Illumina platform (Genewiz Inc.).