Ampure bead

Single cells from the bone marrow (LT-HSC, MkP, CFU-E) were FACS sorted (BD FACS Melody) into 2 μl Smart-seq2 lysis buffer [33 (link)]. Plates were stored at − 80 until further processing according to standard Smart-seq2 protocol [33 (link)]. Reverse transcription conditions: 42 °C for 90 min, 10 cycles 50 °C for 2 min, 42 °C for 2 min, hold at 4 °C; PCR amplification conditions: 98 °C for 3 min, 21 cycles: 98 °C for 20 s, 67 °C for 15 s, 72 °C for 6 min, final extension 72 °C for 5 min, hold at 4 °C. Amplified cDNA underwent bead clean-up in a 1:0.8 × volumetric ratio of Ampure Beads (Beckman Coulter, FL, USA) and eluted into 25 μl of elution buffer (EB, Qiagen, MD, USA). Selected libraries were analyzed on the Bioanalyzer using a High Sensitivity DNA chip (Perkin Elmer, MA, USA) to assess their quality. Next, cDNA was used for library construction using Nextera XT Kit (Illumina, CA, USA). The resulting libraries were pooled in batches of 384 and cleaned up using a 1:0.6 volumetric ratio of Ampure Beads (Beckman Coulter, FL, USA) and eluted into 25 μl of elution buffer (EB, Qiagen, MD, USA). Samples were barcoded during library preparation and 150-bp paired-end reads were generated on a NovaSeq6000 (Illumina, CA, USA).

For whole genome metagenome sequencing, 300 ng of genomic DNA was used after end-repairing (NEBnext ultra II end repair kit, New England Biolabs, MA, United States) and cleaning up with 1× AmPure beads (BeckmannCoulter, United States). The DNA samples were barcoded (LongAmp Taq 2× New England Biolabs, MA, United States) and cleaned up with 1.6× AmPure beads (Beckmann-Coulter, United States). The end-repairing was performed using NEBnext (New England Biolabs, MA, United States) and adapter ligation was performed for 10 min using NEB blunt/TA ligase (New England Biolabs, MA, United States). Library mix was cleaned up using 0.6× AmPure beads and finally eluted in 15 μl of elution buffer. The processed DNA samples were sequenced on GridION X5 (Oxford Nanopore Technologies, Oxford, United Kingdom) using SpotON flow cell (R9.4) in a 48 h sequencing protocol on MinKNOW 2.1 v18.05.5. Nanopore raw reads (“fast5” format) were base called (“fastq5” format) and demultiplexed using Albacore v2.3.1. The reads were compared against NCBI nr database using the diamond tool. The diamond BLASTX alignments were further converted to MEGAN readable format by using the NCBI taxonomy to summarize and order the results. MEGAN GUI is then used to estimate and interactively explore the taxonomical content by checking the read assignment from phylum to species level classification.

End-repairing of bacteriophage HCF1 DNA samples was done using NEBnext ultra II end repair kit (New England Biolabs, Ipswich, MA, United States). Clean-up of end-repaired samples was carried out with 1x AmPure beads (Beckmann Coulter, United States). NEB blunt/TA ligase (New England Biolabs) using NBD103 (ONT) was used for Native barcode ligation. Following ligation, clean-up process was repeated with 1x AmPure beads (Beckmann Coulter). The Qubit-quantified barcode ligated DNA sample was then pooled at equimolar concentrations to obtain 500 ng pooled sample. BAM Adapter ligation was performed for 15 min using NEBnext Quick Ligation Module (New England Biolabs). The library mix was cleaned up using 0.4X AmPure beads (Beckmann Coulter) and, finally, the sequencing library was eluted in 15 μL of elution buffer and used for Nanopore sequencing. Sequencing was performed on a GridION X5 (Oxford Nanopore Technologies, Oxford, United Kingdom) using a SpotON flow cell (R9.4) in a 48 h sequencing protocol on MinKNOW 2.1 v18.05.5. In order to eliminate probable errors in long-read assemblies, all the Nanopore raw reads (“fast5” format) were basecalled (“fastq5” format) and demultiplexed using Albacore v2.3.1. Basecalled reads were error-corrected and assembled using “Canu” assembler v1.8, for sequence polishing.

Solution hybrid selection (SHS) was conducted on FW sample according to the protocol described by Ribière et al. (2016). Briefly, 500 ng of heat denatured libraries was hybridized to the set of biotinylated RNA probes for 24 h at 65°C. Probe/target heterodimers were trapped by streptavidin‐coated paramagnetic beads (Dynabeads M‐280 Steptavidin, Invitrogen, Carlsbag, CA, USA). After several washing steps, the captured targets were eluted from the beads using NaOH and purified using AMPure beads (Beckman Coulter Genomics, Takeley, Essex, United Kingdom). Enriched products were PCR amplified using primers complementary to the library adapters and purified again using AMPure beads (Beckman Coulter Genomics). To increase the enrichment, a second round of hybridization and amplification was performed using the obtained captured products.

Two-week-old Arabidopsis seedlings of Col-0 wild type and trb1/2/3 triple mutans were used for DNA extraction using DNeasy Plant Mini Kit (QIAGEN). A total of 500 ng DNA was sheared with Covaris S2 (Covaris) into around 200 bp at 4 °C. The DNA fragments were used to perform end repair reaction using the Kapa Hyper Prep kit (Roche), and together with TruSeq DNA Sgl Index Set A/B (Illumina) to perform adapter ligation. The ligation products were purified with AMPure beads (Beckman Coulter), and then converted with EpiTect Bisulfite kit (QIAGEN). The converted ligation products were used as templates, together with the primers from the Kapa Hyper Prep kit (Roche) and MyTaq Master mix (Bioline), to perform PCR. The PCR products were purified with AMPure beads (Beckman Coulter) and sequenced by an Illumina NovaSeq 6000 sequencer.

A total of 54 samples, including additional experimental controls, were submitted for 16S rRNA sequencing. The Illumina 16S metagenomic Sequencing Library Preparation protocol was followed. All samples underwent amplicon PCR clean-up using AMPure beads (Beckman Coulter, Indianapolis, IN, USA). Illumina sequencing adapters and dual-index barcodes were added to each library using an Index PCR Illumina XT Index Kit v2 (Illumina Inc., San Diego, CA, USA) followed by PCR clean-up with AMPure beads (Beckman Coulter). Libraries were quantified using the Qubit Fluorometer and the Qubit dsDNA HS Kit (Thermo Fisher Scientific, Waltham, MA, USA). Library fragment-length distributions were analysed using the Agilent TapeStation 4200 and the Agilent D1000 ScreenTape Assay (Agilent Technologies, Santa Clara, CA, USA). Libraries were then pooled in equimolar amounts. Library pool quantification was performed using the KAPA Library Quantification Kit for Illumina (Roche). The library pool was sequenced on an Illumina MiSeq using a MiSeq v2 500 cycle Kit (Illumina; MS-102-2003, San Diego, CA, USA) to generate 250 bp paired-end reads.

Genomic DNA was isolated from cotton leaves, ovules at 0 DPA and 14 DPA, and fibers at 14 DPA using CTAB method [52 (link)]. Total genomic DNA (~5 μg) was fragmented to 100–1000 bp using Bioruptor (Diagenode, Denville, New Jersey). End repair (NEBNext^® End Repair Module) was performed on the DNA fragment by adding an 'A' base to the 3'end (NEBNext^® dA-Tailing Module), and the resulting DNA fragment was ligated to the methylated DNA adapter (NEXTflex^™ DNA Barcodes, Bioo Scientific, Austin, Texas). The adapter-ligated DNA of 200–400 bp was purified using AMPure beads (Beckman Coulter, Brea, California), followed by sodium bisulfite conversion using MethylCode^™ Bisulfite Conversion Kit (Life Technologies, Foster City, California). The bisulfite-converted DNA was amplified by 12 cycles of PCR using LongAmp^®Taq DNA Polymerase (NEB, Ipswich, Massachusetts) and purified using AMPure beads (Beckman Coulter, Brea, California). The Paired-End sequencing of the MethylC-seq libraries was performed for 101 cycles.